Transcription Factors, DNA and Methods for Introduction of Value-Added Seed Traits and Stress Tolerance

ABSTRACT

Abscisic acid-inducible gene expression in different plant tissues is enhanced synergistically by the co-expression of a B3-domain transcription factor and various bZIP-domain transcription factors, or a different B3-domain transcription factor. Using these transcription factors in novel formulations, as shown by examples, will confer value-added traits to transgenic plants, including, but not limited to, higher levels of heterologous gene expression, drought and salt tolerance, viability and productivity under stress, and enhanced nutrient reserves and seed properties.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of pending U.S. application Ser. No.10/996,058 filed Nov. 24, 2004, the contents of which are incorporatedherein by reference. This is a continuation-in-part of pending U.S.application Ser. No. 10/629,907, filed Jul. 30, 2003, which is basedupon Provisional Application No. 60/399,565 filed Jul. 30, 2002, both ofwhich are incorporated fully herein by reference

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file namedrocksequence.txt, created on Jul. 30, 2003, and having a size of 173 KBand is filed concurrently with the specification. The sequence listingcontained in this ASCII formatted document is part of the specificationand is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

A variety of stress-related traits in plants are enhanced by thesynergistic effects on abscisic acid (ABA)-inducible gene expression ofco-expressed basic leucine zipper (bZIP)-domain transcription factorsand B3-domain transcription factors. Additionally, two differentB3-domain transcription factors may be used to synergistically regulateABA-inducible gene expression.

2. Description of Related Art

The growth in the world's population combined with a general increase inglobal prosperity is creating an increasing demand for food, fiber andsustainable agriculture. It is estimated that the world's populationwill increase by 80% to 10.8 billion people by 2050, with a concomitantdecrease in arable land of 20%. A worthwhile future can only beguaranteed through sustainable agriculture and a protective relationshipwith nature. For example, rice is the staple food for two-thirds of theworld's population and is the primary cereal crop in the world, withworldwide production in 2000 of 600 million tons. 92% of the world'srice is produced in Asia, and 40% of the cultivated area is rain-fed andexperiences environmental stress, with losses estimated at 200 milliontons/yr. Another example is impacts of drought on maize production inthe southwest United States. Based on data from the USDA NationalAgricultural Statistics Service (http://www.usda.gov/nass/), in 2003 thegeographical region spanning southern CA, AZ, NM, TX High Plains andtrans-Pecos regions produced 1.2% (127 mil bushels) of the nation'sgrain corn, valued at >$310 million. It is noteworthy that only 78% ofgrain corn planted in these regions of the Southwest were subsequentlyharvested, whereas on average 90% of the planted acreage was harvestedthroughout the United States. Significantly, for AZ and NM from1999-2003, the percentages of grain corn harvested ranged from only35-58%, while the national average over that time period was 90.2%. Thebasis for these differences between the southwest and the “Corn Belt”harvests, valued at >$48 million in losses last year alone, is due toreductions in yield due to drought stress typically experienced by cropsin the southwest. Genetic engineering of maize for increased vegetativedrought stress adaptation should result in increased yields and profitsfor producers. A third example is dryland cotton; estimates of thevalue-added worth of cotton with increased photosynthetic and water useefficiencies and improved seed qualities exceed $200 million/yr in westTexas, $1 billion/yr in the USA, and $5 billion/yr globally.

Yield enhancement to increase crop production is one of the essentialstrategies to meet the demand for food by the growing population. Inorder to supply the world's population in 20 years' time with enough toeat, today's food production will have to be doubled on a third lessland and water. For example, due to traditional rice breeding advances,with which germplasm from wild relatives was transferred to cultivatedstrains, production of rice doubled between 1966 and 1990, but it isestimated that production must increase 60% by 2025 to meet demand. Therate at which growers have been able to further improve cropproductivity has declined as improved farming practices have become morefully implemented around the world, as land in developed countriesavailable for conversion to farming has declined, and as concerns aboutthe environmental impact of farming have increased.

While the rate of yield increases from hybrids has slowed in the lasttwo decades, the application of biotechnology and genomics isdramatically increasing innovation in the agricultural and seedindustries. Biotechnology as a means of sustainable agriculture is acrucial component to meeting the challenges posed by the interrelatedglobal issues of poverty, hunger, population growth, and environmentaldegradation in the twenty-first century. Biotechnology enablesgene-by-gene analysis and enhancement of crops and is augmentingtraditional breeding by enabling faster, targeted development ofperformance-enhancing traits. These traits currently are designed tocreate higher-quality animal feed and in the future are expected toinclude nutritional benefits for humans.

Growers have rapidly adopted the first generation of geneticallyengineered seed traits, with significant numbers of acres planted. Thenumber of global planted acres of herbicide-tolerant andinsect-resistant crops grew from less than 5 million acres in 1995 toapproximately 120 million acres in 1999. Despite this rapid growth, thetotal number of acres covered currently represents only a small fractionof the approximately 3 billion acres of crops cultivated worldwide.Additional growth will come from further adoption of currently availabletraits and the development of new input and output traits.

Improvement of crop plants for a variety of traits, including diseaseand pest resistance, adaptation to abiotic stresses, and grain qualityimprovements such as oil, starch or protein composition, has beenachieved by introducing new or modified genes into plant genomes. It hasrecently been shown that the “Green Revolution” of the 1960s thatresulted in large increases in wheat yields was due to adoption ofvarieties that contain a dominant allele of a gene that controlstranscription factor expression by modulating microRNAs, a newlydiscovered mechanism of gene regulation in mesozoans. Transcriptionfactors control virtually all significant plant traits, including yield,disease resistance, freezing and drought protection, as well as theproduction of chemicals and proteins used as pharmaceuticals,nutriceuticals and consumer products, by coordinate regulation ofmultiple target genes whose functions in many cases are not yet known.

The expression of target transgenes and endogenous genes is controlledthrough a complex set of protein/DNA and protein/protein interactions.Promoters and enhancers can impart patterns of expression that areeither constitutive or limited to specific tissues or times duringdevelopment, or in response to environmental stimuli. There arelimitations in the types of expression achievable using existingpromoters for transgene expression. One limitation is in the expressionlevel achievable. It is difficult to obtain traits that requirerelatively high expression of an introduced gene, due to limitations inpromoter strength. A second limitation is that the pattern of expressionconferred by the particular promoter employed is inflexible in that thesame promoter-dependent pattern of expression is conferred fromgeneration to generation. It is desirable to have the ability toregulate trait-conferring transgene expression differently in successivegenerations. One example would be a trait that has a side effect ofbeing detrimental to seed quality, but which is desired for use infodder. In this case, it would be desirable to carry thetrait-conferring transgene in an inactive state in separate breedingstocks.

Plants are sessile and therefore must perpetually develop in response totheir changing environment. Plants have evolved complex, integrated, andoverlapping signaling pathways to maintain a plastic growth habit inresponse to stresses such as drought, salt, cold, as well as hormonalcues such as abscisic acid (ABA). ABA mediates a myriad of physiologicalprocesses in growth and development, including cell division, water useefficiency, and gene expression during seed development and in responseto environmental stresses such as drought, chilling, salt, pathogenattack, and UV light. Despite the complex multitude of physiological,molecular, genetic, biochemical, and pharmacological data that implicateABA in stress responses, the adaptive responses to ABA and stresses, andthe pathways that trigger them, are largely unknown. Seed maturation andfreezing/drought/salt tolerance may have certain protective mechanismsin common, since they share the common phenomenon of dehydration stress.

It would be advantageous for genetic engineering of plants withenvironmental stress resistance to regulate multiple genes in aparticular metabolic or response pathway via a single transgene. Cloningand overexpression of Drought Response Element Binding (DREB)/ColdBinding Factor (CBF) subfamily of the AP2-domain family of transcriptionfactors responsible for cold-inducible gene expression has demonstratedthe practical benefits of coordinated activation of uncharacterized genesets that can confer non-specific protection to transgenic plants byup-regulation or pre-activation of stress-response pathways. TheABA-INSENSITIVE-4 gene (ABI4) is most closely related to the DREB/CBFsubfamily of the AP2-domain family. Transgenic overexpression of thetranscription factor ALFIN1 enhances expression of the endogenous MsPRP2gene in alfalfa and improves salinity tolerance of the plants.Over-expression of a single Ca²⁺-dependent protein kinase confers bothcold and salt/drought tolerance on rice plants. A multi-componenttranscription factor/target promoter system that regulates hormone andstress responses could be used to address the limitations of singletransgene expression and tap into the natural defense systems of cropplants.

Although hundreds of ABA-regulated genes have been identified to date,many of them homologs from a broad range of species, these are likely torepresent a somewhat anecdotal sampling of the full spectrum ofABA-responsive genes. Preliminary genome profiling in Arabidopsis hasallowed estimation of the number of plant genes modulated by ABA, withcurrent estimates at about 2000-6000 genes. A number of plant geneproducts have been identified that may function in desiccationtolerance. The COR genes are cold-, drought-, salt-, and ABA-responsivegenes whose protein products are heat stable and hydrophilic; some CORgenes have structural similarities to the late embryogenesis-abundant(LEA) proteins. LEA homologues in wheat, maize, barley, carrot, and theresurrection plant Craterostigma plantagineum are induced by ABA anddehydration stress. The exact roles of COR and LEA genes in cold anddesiccation tolerance are not yet known, but there is strong evidencethat they have an adaptive function in desiccation, freezing, and salttolerance. Altered expression of ABA signal transduction genes can havebeneficial effects on stress adaptation of plants.

The RY-G-box-RY regulatory element is commonly found in seed storageprotein gene promoters and is necessary for seed-specific expression ofthe β-phaseolin and Em promoters. The sequences of the Ry-G-box-RYelements that are found in different natural promoters have variations,but can be recognized by the presence of particular nucleotidesequences: CATGCAW (the “RY” feature) and CACGTG (the “G-box”). There issubstantial diversity in the cis sequences shown to confer ABA-inducibleexpression. The smallest promoter units (called ABA-Response Elements;ABREs) that are both necessary and sufficient for ABA induction of geneexpression appear to consist of at least two essential cis elements, oneof which is usually a G-box and the other a “coupling” element.

A seed-specific regulatory factor, Viviparous-1 (VP1), was firstdescribed in 1931 and was cloned by transposon tagging in 1989. TheABA-INSENSITIVE3 (ABI3) gene of Arabidopsis is the genetic equivalent ofmaize VP1 and was cloned by chromosome walking. VP1/ABI3 is expressed indeveloping seeds and precedes ABA-inducible storage protein andlate-embryogenesis-abundant (LEA) gene expression. Rice and maizeprotoplasts that transiently overexpress the VP1 cDNA can transactivateABA-inducible promoters from numerous species. Similar transactivationresults have been obtained in homologous transient gene expressionsystems with the rice VP1 and bean Pv-ALF orthologs. Remarkably, VP1also has repressor activity towards the germination-specificalpha-amylase genes, but repression is non-cell-autonomous and requiresembryo-specific factors other than ABA and VP1.

Structure/function studies with VP1 and PvALF in transient geneexpression assays demonstrate that the highly conserved N-terminalacidic domain (A1, VP1 amino acids [aa] 51-163) functions as atranscriptional activator and acts in synergy with ABA. The acidicdomain of VP1 is not required for germination-specific alpha-amylasegene repression. The conserved basic B2 region (aa 508-544 of VP1) isrequired for transactivation of the ABA-inducible Em promoter and forenhancing the in vitro binding of various basic leucine zipper (bZIP)factors to their cognate targets, but not for alpha-amylase generepression. The B3 domain (aa 632-760) binds specifically to promotersequences required for transactivation but not to ABA-responsivecis-elements. Furthermore, the B3 domain is not required for synergisticeffects of transactivation with ABA or for alpha-amylase generepression. Pv-ALF facilitates chromatin modification of theABA-inducible β-Phas promoter, which in turn potentiates ABA-mediatedtranscription. Carrot and Arabidopsis Pv-ALF orthologs can also directABA-inducible seed storage protein expression in leaves when expressedectopically. The exact molecular mechanisms of ABI3/VP1/Pv-ALF are notknown, but the predicted FUS3 and LEAFY COTYLEDON-2 class of regulatorsthat control embryo maturation have a continuous stretch of more than100 amino acids showing significant sequence similarity to the conservedB3 domains of ABI3/VP1/Pv-ALF. Taken together with genetic results thatshow FUSCA3 and LEC2 interact with ABI3, these correlations suggest thatABI3, LEC2, and FUS3 may act in partially redundant pathways. TheArabidopsis genome encodes 43 members of the B3-domain family, 19 ofthem within the ABI3/VP1-related subfamily, and their functions arelargely unknown.

There are 81 predicted bZIP factor genes in Arabidopsis, but only onebZIP subfamily has been genetically or functionally linked to ABAresponse: that composed of ABI5 and its homologs, including the ABREBinding Factors (ABFs and AREBs), Enhanced Em Level (EEL/AtbZIP12), andAtbZIP13-15, 27, and 67, which include the AtDPBFs (Arabidopsis thalianaDc3 Promoter Binding Factors). Homologs of these genes have beencharacterized in sunflower and rice, where they are also correlated withABA-, seed- or stress-induced gene expression. However, studies of bZIPsfrom other species have shown that in vitro binding of ABREs need notreflect action in ABA signaling in vivo. A rice homolog of ABI5, TRAB1,was identified by a yeast two-hybrid screen using the basic domains ofOsVP1 as “bait” and shown to interact with ABREs in vitro and activateABA-inducible transcription in rice protoplasts. AREBs and ABFs bothshare with ABI5 three conserved charged domains (C1-C3) in theiramino-halves as well as the bZIP domain and another conserved (C4)domain at the C-terminus. In vitro studies with the DPBFs and otherABI5-family members have demonstrated that this subfamily binds to G-boxpromoter elements (ABREs) required for ABA regulation. However, theABI5/DPBF/ABF/AREB subfamily has a broader consensus sequence for itsbinding site than the other bZIP proteins in that its members toleratevariability in the ACGT core element essential to the ABRE G-box. ABI5and its homolog DPBF4/EEL were shown to compete for the same bindingsites in the AtEM1 promoter and a model was proposed, based on singleand double mutant phenotypes of altered gene expression, that EELdirectly antagonized ABI5 transactivation. Analyses of transcriptaccumulation in abi5 mutants suggest that, similar to ABI3, ABI5 hasboth activator and repressor functions, but that ABI5 and ABI3 may haveeither synergistic or antagonistic effects on gene expression, dependingon the gene. ABI5 protein accumulation is further enhanced byABA-induced phosphorylation and resulting stabilization of the protein,at least during the early phases of germination.

As indicated above, it has been previously shown that maize VP1 isfunctionally redundant with ABI3 and that other orthologues of VP1/ABI3could substitute for VP1 in a multi-component heterologoustransactivation system. Consistent with this, expression of anArabidopsis GIBBERELLIN-INSENSITIVE (GAI) orthologue (the same generesponsible for the “Green Revolution” in wheat; see above) intransgenic rice resulted in desirable dwarfing traits, suggesting thatheterologous regulatory genes can be used to affect traits in a widerange of crop species. Transgenic rice plants that express the maizephosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphatedikinase (PPDK) exhibit a higher photosynthetic capacity (up to 35%)than untransformed plants, mainly associated with an enhanced stomatalconductance and a higher internal CO₂ concentration. An additionalbenefit of using heterologous genes is that they may minimize artifactssuch as co-suppression and posttranscriptional transgene silencing.

Coordinated regulation of multiple endogenous genes is important forstress adaptation. New methods which genetically engineer value-addedvegetative traits for stress adaptation and seed qualities by directedexpression of ABA-related transcription factors would be beneficial tosupply the world with the increased amounts of food needed by futuregenerations. By overcoming the limitations of targeted gene expressionand by transactivation of endogenous plant stress adaptation pathways,the volume and quality of plant products, especially from environmentsunder stress, will be improved.

BRIEF SUMMARY OF THE INVENTION

Transgenic plants that ectopically express a B3-domain transcriptionfactor, preferably VP1, ABI3, RAV2 and homologues from various species,in combination with at least one different B3-domain transcriptionfactor or a bZIP-domain transcription factor, preferably ABI5 andABI5-like family members ABF1 and ABF3 and homologues from variousspecies, will respond to abiotic stressors such as salt, drought, andcold by activating ABA-inducible target promoters as well as endogenouspromoters that coordinate expression of genes involved in stressadaptation, such as LEA and COR genes. In preferred embodiments, theB3-domain transcription factor and the bZIP-domain transcription factorare those found in members of the genus Arabidopsis and Zea mays. Thismulti-component expression system may be expanded by modifying theregulatory sequences of the target promoter or the promoter driving thetranscription factor effectors to include tissue-specific enhancerelements or stress-response elements to further direct the expression ofthe target gene of interest. It is known in animal systems thattargeting of some combinations of transcription factors to the samepromoter may produce synergistic effects on the expression level. Thestrategy disclosed herein amplifies the expression level from anABA-inducible promoter. For example, recent results have shown thatgrain-filling in rice is critically dependent on water status and ABAlevels, suggesting that amplification of ABA response pathways byectopic transcription factor expression in appropriate tissues and atcritical times during development could have beneficial effects. Themulti-component benefits of transcription factor synergy may be realizedby genetic crossing of two lines harboring separate transcription factorcomponents.

Abscisic acid (ABA) signaling pathways are highly conserved amongmonocots and dicots (See Gampala et al., J. Biol. Chem. 277: 1689(2002)). Therefore, a multi-component transgenic approach to engineeringstress tolerance with effectors from diverse plant species is practical.Evidence presented herein with overexpressed Arabidopsis bZIP domaintranscription factors called ABFs, AREB3, DPBF4/EEL, ABI5 and maize B3domain transcription factor VP1 in rice embryonic and maize mesophyllprotoplasts further extends this claim. Since it has been shown thatmaize VP1 is functionally redundant with ABI3, other orthologues ofVP1/ABI3 from various species could substitute for VP1 in amulti-component heterologous transactivation system. Further, evidenceis presented herein that a VP1/ABI3-related B3 domain homologue fromArabidopsis, RAV2, functions in synergy with VP1 and the bZIPs ABI5 andABF3 in maize mesophyll protoplasts, demonstrating a novel combinatorialphenomenon of synergy that extends the scope of this invention. Onepotential drawback to overexpressing regulatory factors that conferstress tolerance to transgenic crops is reduced yields throughpleiotropic “knock on” effects that indeed may be the direct consequenceof stress adaptation mechanisms triggered by the transgene effector. Inthis scenario, the present invention would still find application inhorticultural plants like turf grasses where yields, per se, may be lessimportant. Likewise, in ornamental species the slow-growth,stress-adapted phenotype would be a value-added trait. Some novelactivities of ABI5-like family members and ABI5 alone and in combinationwith maize VP1 have been demonstrated. Ectopic or controlled, e.g.inducible, expression of these and related effectors in any plantspecies should result in conditionally altered stress responses andhigher levels of engineered target gene expression than otherwisepossible. The present invention also relates to polynucleotides whichcontain the complete gene with the polynucleotide sequence correspondingto SEQ ID NO:.1, SEQ ID NO.:3, or SEQ ID NO: 5 or fragments thereof, andwhich can be obtained by screening by means of the hybridization of acorresponding gene bank with a probe which contains the sequence of saidpolynucleotide corresponding to SEQ ID NO:.1, SEQ ID NO.:3, or SEQ IDNO: 5 or a fragment thereof, and isolation of said DNA sequence.

Given the efficacy of ABA in regulating such basic processes as seeddevelopment, dormancy vs. germination, transpiration and stressresponses, the present invention can pave the way to importantbiotechnological applications.

BRIEF DESCRIPTION OF THE DRAWINGS

The features and advantages of the present invention will becomeapparent from the following detailed description of a preferredembodiment thereof, taken in conjunction with the accompanying drawings,in which:

FIG. 1. depicts two graphs showing that A) the overexpression of ABF3and ABF4 in rice protoplasts is sufficient to transactivateABA-inducible Em-GUS expression; and B) that overexpressed ABF3interacts synergistically with ABA and VP1, but not ABI5.

FIG. 2 depicts two graphs showing that A) ABI5, ABF3, ABF4, AREB3, orDPBF4/EEL overexpression in maize mesophyll protoplasts is sufficient totransactivate Em-GUS expression, while B) ABI5, ABF1, and ABF3 but notABF2, AREB3, ABF4, or DPBF4/EEL can synergize with co-transformed VP1;and

FIG. 3 is a graph in two parts showing that A) RAV2 interactssynergistically with the bZIP transcription factors ABI5 and ABF3; andB) that RAV2 interacts synergistically with VP1 in maize mesophyllprotoplasts.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one of ordinary skill inthe art of the present invention. It is to be understood that thisinvention is not limited to the particular methodology, protocol, andreagents described, as these may vary.

All publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 25% sequenceidentity. Alternatively, percent identity can be any integer from 25% to100%. More preferred embodiments include at least: 39%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a referencesequence using the programs described herein; preferably BLAST usingstandard parameters, as described below.

Accordingly, “B3-domain transcription factors” are transcription factorsthat include a polynucleotide sequence having a B3 domain which is atleast 25% identical to the B3 domain of SEQ ID NO:1. More preferably,the B3-domain transcription factor is a transcription factor thatincludes a polynucleotide sequence having a B3 domain which is at least45% identical to the B3 domain of SEQ.ID.NO.: 1. Additionally, theB3-domain transcription factors are transcription factors that include apolypeptide sequence having a B3 domain which is at least 40% identicalto the B3 domain of SEQ ID NO:2. More preferably, the B3-domaintranscription factor is a transcription factor that includes apolypeptide sequence having a B3 domain which is at least 50% identicalto the B3 domain of SEQ.ID.NO.: 2. The B3-domain transcription factorsalso include transcription factors that include a polynucleotidesequence having a B3 domain which is at least 25% identical to the B3domain of SEQ ID NO:3. More preferably, the B3-domain transcriptionfactor is a transcription factor that includes a polynucleotide sequencehaving a B3 domain which is at least 45% identical to the B3 domain ofSEQ.ID.NO.: 3. Additionally, the B3-domain transcription factors includetranscription factors that include a polypeptide sequence having a B3domain which is at least 40% identical to the B3 domain of SEQ ID NO: 4.More preferably, the B3-domain transcription factor is a transcriptionfactor that includes a polypeptide sequence having a B3 domain which isat least 50% identical to the B3 domain of SEQ.ID.NO.: 4. The “bZIPdomain transcription factors” include transcription factors that includea polynucleotide sequence having a bZIP domain which is at least 25%identical to the bZIP domain of SEQ ID NO:5. More preferably, the bZIPdomain transcription factor is a transcription factor that includes apolynucleotide sequence having a bZIP domain which is at least 45%identical to the bZIP domain of SEQ.ID.NO.: 5. Additionally, the bZIPdomain transcription factors include transcription factors that includea polypeptide sequence having a bZIP domain that is at least 40%identical to the bZIP domain of SEQ ID NO: 6. More preferably, the bZIPdomain transcription factor is a transcription factor that includes apolypeptide sequence having a bZIP domain which is at least 60%identical to the bZIP domain of SEQ.ID.NO.: 6. One of skill willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like. Substantial identity of amino acidsequences for these purposes normally means sequence identity of atleast 60%. Preferred percent identity of polypeptides can be any integerfrom 40% to 100%. More preferred embodiments include at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Most preferredembodiments include at least 60% polypeptide identity.

The phrase “nucleic acid” refers to a single or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Nucleic acids may also include modified nucleotides that permitcorrect read through by a polymerase and do not alter expression of apolypeptide encoded by that nucleic acid.

The phrase “polynucleotide sequence” or “nucleic acid sequence” includesboth the sense and antisense strands of a nucleic acid as eitherindividual single strands or in the duplex. It includes, but is notlimited to, self-replicating plasmids, chromosomal sequences, andpharmacological application of polymers of DNA or RNA. For example, see“Therapeutic silencing of an endogenous gene by systemic administrationof modified siRNAs” Nature 432: 173-178 (Nov. 11, 2004). The presentinvention also includes small interfering RNAs (siRNAs) homologous tothe bZIP domain transcription factors and B3 domain transcriptionfactors.

The phrase “nucleic acid sequence encoding” refers to a nucleic acidwhich directs the expression of a specific protein or peptide. Thenucleic acid sequences include, but are not limited to, both the DNAstrand sequence that is transcribed into RNA and the RNA sequence thatis translated into protein. The nucleic acid sequences include both thefull length nucleic acid sequences as well as non-full length sequencesderived from the full length sequences. It should be further understoodthat the sequence includes the degenerate codons of the native sequenceor sequences which may be introduced to provide codon preference in aspecific host cell.

A polynucleotide “exogenous to” an individual plant is a polynucleotidewhich is introduced into the plant by any means other than by a sexualcross. Examples of means by which this can be accomplished are describedbelow, and include Agrobacterium-mediated transformation, biolisticmethods, electroporation, in planta techniques, and the like. Such aplant containing the exogenous nucleic acid is referred to here as an R1generation transgenic plant. Transgenic plants which arise from sexualcross or by selfing are descendants of such a plant.

A “B3 domain polynucleotide” is a nucleic acid sequence comprising acoding region of about 100 to about 900 nucleotides, sometimes fromabout 200 to about 630 nucleotides, which hybridizes to SEQ ID NO:1 or 3under stringent conditions, preferably as defined below, or whichencodes a B3 domain polypeptide. B3 domain polynucleotides can also beidentified by their ability to hybridize under low stringency conditions(e.g., Tm about 40° C.) to nucleic acid probes having a sequence fromposition 559 to 885 in SEQ ID NO:3 or from position 1560 to 1929 in SEQID NO:1.

A “B3 domain polypeptide” is a sequence of about 100 to about 130 aminoacid residues encoded by a B3 domain polynucleotide. A full length B3domain polypeptide can act as a subunit of a protein capable of actingas a transcription factor in plant cells.

A “bZIP domain polynucleotide” is a nucleic acid sequence comprising acoding region of about 190 nucleotides which hybridizes to SEQ ID NO:5under stringent conditions, preferably as defined below, or whichencodes a bZIP domain polypeptide. bZIP domain polynucleotides can alsobe identified by their ability to hybridize under low stringencyconditions (e.g., Tm about 40° C.) to nucleic acid probes having asequence from position 1057 to 1251 in SEQ ID NO:5.

A “bZIP domain polypeptide” is a sequence of about 60 amino acidresidues encoded by a bZIP domain polynucleotide. A full length bZIPdomain polypeptide can act as a subunit of a protein capable of actingas a transcription factor in plant cells.

As used herein, a “homolog” of a particular gene (e.g., SEQ ID NO:1) isa second gene in the same plant type or in a different plant type, whichhas a polynucleotide sequence of at least 50 contiguous nucleotideswhich are substantially identical (determined as described herein) to asequence in the first gene. It is believed that, in general, homologsshare a common evolutionary past.

“Increased or enhanced B3 domain activity or expression of a B3 domaingene” refers to an augmented change in B3 domain activity. Examples ofsuch increased activity or expression include the following. B3 domainactivity or expression of the B3 domain gene is increased above thelevel of that in wild-type, non-transgenic control plants (i.e. thequantity of B3 domain activity or expression of the B3 domain gene isincreased). B3 domain activity or expression of the B3 domain gene is inan organ, tissue or cell where it is not normally detected in wild-type,non-transgenic control plants (i.e. spatial distribution of B3 domainactivity or expression of the B3 domain gene is increased). B3 domainactivity or expression is increased when B3 domain activity orexpression of the B3 domain gene is present in an organ, tissue or cellfor a longer period than in a wild-type, non-transgenic controls (i.e.duration of B3 domain activity or expression of the B3 domain gene isincreased).

“Increased or enhanced bZIP domain activity or expression of a bZIPdomain gene” refers to an augmented change in bZIP domain activity.Examples of such increased activity or expression include the following.bZIP domain activity or expression of the bZIP domain gene is increasedabove the level of that in wild-type, non-transgenic control plants(i.e. the quantity of bZIP domain activity or expression of the bZIPdomain gene is increased). bZIP domain activity or expression of thebZIP domain gene is in an organ, tissue or cell where it is not normallydetected in wild-type, non-transgenic control plants (i.e. spatialdistribution of bZIP domain activity or expression of the bZIP domaingene is increased). bZIP domain activity or expression is increased whenbZIP domain activity or expression of the bZIP domain gene is present inan organ, tissue or cell for a longer period than in a wild-type,non-transgenic controls (i.e. duration of bZIP domain activity orexpression of the bZIP domain gene is increased).

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe sequence is complementary to all or a portion of a referencepolynucleotide sequence.

The term “isolated” means separated from its natural environment.

The term “polynucleotide” refers in general to polyribonucleotides andpolydeoxyribonucleotides, and can denote an unmodified RNA or DNA or amodified RNA or DNA.

The term “polypeptides” is to be understood to mean peptides or proteinswhich contain two or more amino acids which are bound via peptide bonds.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing).

Polypeptides which are “substantially similar” share sequences as notedabove except that residue positions which are not identical may differby conservative amino acid changes. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

An “isolated polynucleotide” or an “isolated DNA segment” having asequence which encodes a plant transcription factor is a polynucleotidewhich contains the coding sequence of the plant transcription factor (i)in isolation, (ii) in combination with additional coding sequences, suchas fusion protein or signal peptide, in which the plant transcriptionfactor coding sequence is the dominant coding sequence, (iii) incombination with non-coding sequences, such as control elements, such aspromoter and terminator elements, effective for expression of the codingsequence in plant cells, and/or (iv) in a vector or host environment inwhich the plant transcription factor coding sequence is a heterologousgene.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in many bacteria andsome eukaryotes.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a distal gene. The promoterwill generally be appropriate to the host cell in which the target geneis being expressed. The promoter together with other transcriptional andtranslational regulatory nucleic acid sequences (also termed “controlsequences”) is necessary to express a given gene. In general, thetranscriptional and translational regulatory sequences include, but arenot limited to, promoter sequences, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, enhancer or activator sequences, and regulatory small RNAtarget-binding sequences. The term “promoter” also refers to a region orsequence determinants located upstream or downstream from the start oftranscription and which are involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Such promoters need not be of plant origin, for example,promoters derived from plant viruses, such as the CaMV35S promoter, canbe used in the present invention. As used herein, the term “promoter”refers to a sequence of DNA that functions to direct transcription of agene which is operably linked thereto. A promoter may or may not includeadditional control sequences (also termed “transcriptional andtranslational regulatory sequences”), involved in expression of a givengene product. In general, transcriptional and translational regulatorysequences include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, enhancer or activator sequences, andregulatory small RNA target-binding sequences. The promoter may behomologous or heterologous to the cell in which it is found.

A nucleic acid sequence is “heterologous” with respect to a controlsequence (i.e. promoter or enhancer) when it does not function in natureto regulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid constructs areintroduced into the cell or part of the genome in which they arepresent, and have been added to the cell by transfection,microinjection, electroporation, or the like. The sequences may containa control sequence/DNA coding sequence combination that is the same as,or different from, a control sequence/DNA coding sequence combinationfound in the native plant. A polynucleotide sequence is “heterologousto” an organism or a second polynucleotide sequence if it originatesfrom a foreign species, or, if from the same species, is modified fromits original form. For example, a promoter operably linked to aheterologous coding sequence refers to a coding sequence from a speciesdifferent from that from which the promoter was derived, or, if from thesame species, a coding sequence which is different from any naturallyoccurring allelic variants.

As used herein, the term “operably linked” relative to a recombinant DNAconstruct or vector means nucleotide components of the recombinant DNAconstruct or vector are in a functional relationship with anothernucleic acid sequence. For example, a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons). The term “gene” may be used interchangeably herein with theterm “nucleic acid coding sequence”, and the term “structural gene”which means a DNA coding region.

As used herein, the term “fragment,” when referring to a gene sequencemeans a polynucleotide having a nucleic acid sequence which is the sameas part of, but not all of, the nucleic acid sequence of the full lengthgene. The fragment preferably includes at least 15 contiguous bases ofthe gene, preferably at least 20-30 bases. With reference to interactionwith a transcription factor, the sequence must be of sufficient lengthto interact with the transcription factor.

As used herein, the terms “transformed”, “stably transformed” or“transgenic” with reference to a plant cell means the plant cell has anon-native (heterologous) nucleic acid sequence integrated into itsgenome which is maintained through one or more generations.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process generally includes both transcription, and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, the term “effector” refers to plant transcriptionfactors that “effect” the transcription of genes having the appropriateresponse sequence.

As used herein, the terms “regulatable promoter” and “induciblepromoter” may be used interchangeably and refer to any promoter theactivity of which is affected by a cis- or trans-acting factor.

As used herein, the terms “transcriptional regulatory protein”,“transcriptional regulatory factor” and “transcription factor” may beused interchangeably and refer to a cytoplasmic or nuclear protein thatbinds a DNA response element and thereby transcriptionally regulates theexpression of an associated gene or genes. Transcription factorsgenerally bind directly to a DNA response sequence or element, howeverin some cases may bind indirectly to another protein, which in turnbinds to or is bound to the DNA response element.

As used herein, the terms “response sequence” and “response element”refer to the binding site or sequence for a transcriptional regulatoryprotein (transcription factor) which may be the part of, overlapping, oradjacent to, a promoter sequence.

As used herein, a “plant cell” refers to any cell derived from a plant,including undifferentiated tissue (e.g., callus) as well as plant seeds,pollen, progagules and embryos.

As used herein, the term “mature plant” refers to a fully differentiatedplant.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature.

As used herein, the term “plant” includes reference to whole plants,shoot vegetative organs/structures (for example, leaves, stems, tubers,etc.), roots, flowers and floral organs/structures (e.g. bracts, sepals,petals, stamens, carpels, anthers and ovules), seed (including embryo,endosperm, and seed coat) and fruit (the mature ovary), plant tissue(e.g. vascular tissue, ground tissue, and the like) and cells (e.g.guard cells, egg cells, trichomes and the like), and progeny of thesame. Plant cell, as used herein includes, without limitation, seeds,suspension cultures, embryos, meristematic regions, callus tissue,leaves roots shoots, gametophytes, sporophytes, pollen, and microspores.The class of plants which can be used in the methods of the presentinvention is generally as broad as the class of higher and lower plantsamenable to transformation techniques, including angiosperms (bothmonocotyledenous and dicotyledenous plants), gymnosperms, ferns, andmulticellular algae. It includes plants of a variety of ploidy levels,including aneuploid, polyploid, diploid, haploid and hemizygous.

As used herein, the term “transgenic plant” refers to a plant comprisingwithin its genome a heterologous DNA segment. Generally, theheterologous polynucleotide is stably integrated within the genome suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic.

As used herein, the term “enhancement” means increasing theintracellular activity of one or more enzymes in a plant cell and/orplant which are encoded by the corresponding DNA. Enhancement can beachieved with the aid of various manipulations of the cell. In order toachieve enhancement, particularly over-expression, the number of copiesof the corresponding gene can be increased, a strong promoter can beused, or the promoter and regulation region or the ribosome binding sitewhich is situated upstream of the structural gene can be mutated.Expression cassettes which are incorporated upstream of the structuralgene act in the same manner. In addition, it is possible to increaseexpression by employing inducible promoters. A gene can also be usedwhich encodes a corresponding enzyme with a high activity. Expressioncan also be improved by measures for extending the life of the mRNA.Furthermore, enzyme activity as a whole is increased by preventing thedegradation of the enzyme. Moreover, these measures can optionally becombined in any desired manner. These and other methods for alteringgene activity in a plant are known as described, for example, in Methodsin Plant Molecular Biology, Maliga et al, Eds., Cold Spring HarborLaboratory Press, New York (1995).

Method and Composition of the Invention

The invention provides transgenic plant cells and transgenic plantswhich express at least two transcription factors that interactsynergistically: 1) a first B3-domain transcription factor and 2) asecond B3-domain transcription factor or a bZIP domain transcriptionfactor. Expression of the transcription factors is correlated withincreased expression of a gene under the control of a promoter withwhich the transcription factors interact. The two transcription factorsare expressed in the same cell and act in concert to modulate expressionof the gene to which they are operably linked. Expression of twotranscription factors in the same plant results in a level of transgeneexpression which is greater than the expression of each transcriptionfactor alone, when the transgene is under the control of a promoter withwhich the transcription factors interact. In other words, as exemplifiedherein, the level of expression observed when a transgene is expressedunder the control of a promoter with which both 1) the B3-domaintranscription factor and 2) the bZIP and/or B3-domain transcriptionfactors interact is greater than the expression level observed due tothe additive effects of each individual transcription factor.

The invention provides expression cassettes comprising a promoteroperably linked to a heterologous polynucleotide sequence or complementthereof, encoding a B3 or bZIP domain-containing polypeptide comprisinga sequence which is at least 50% identical to the B3 domain of SEQ IDNO:2 or SEQ ID NO:4 and 60% identical to SEQ ID NO 6. In someembodiments, the polynucleotide sequence is heterologous to any elementin the expression cassette. In a preferred embodiment, the B3 domaincomprises a polypeptide between about amino acid residue 496 and aminoacid residue 619 of SEQ ID NO:2. In a more preferred embodiment, the B3domain comprises a polypeptide sequence between about amino acid residue187 and amino acid residue 295 of SEQ ID NO:4. In yet another preferredembodiment, the bZIP domain comprises a polypeptide between about aminoacid residue 353 and amino acid residue 417 of SEQ. ID NO: 6.

In particularly preferred embodiments, the B3 domain polypeptide isshown in SEQ ID NO:2, 4, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64 or 66. Such B3 domain polypeptides can be encodedby the polynucleotide sequences shown in SEQ ID NO:1, 3, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 or SEQ ID NO:65,respectively. In another embodiment the B3 domain polypeptide is afusion between two or more B3 domain polypeptides or polypeptidesubsequences. The polynucleotide sequence can be heterologous to anyelement in the expression cassette. Such expression cassettes can encodefusions of two or more B3 domain polypeptides or polypeptidesubsequences.

In particularly preferred embodiments, the bZIP domain polypeptide isshown in SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28. SuchbZIP domain polypeptides can be encoded by the polynucleotide sequencesshown in SEQ ID NO: 5, 7, 9 11, 13, 15, 17, 19, 21, 23, 25 or 27. Inanother embodiment the bZIP domain polypeptide is a fusion between twoor more bZIP domain polypeptides or polypeptide subsequences. Thepolynucleotide sequence can be heterologous to any element in theexpression cassette. Such expression cassettes can encode fusions of twoor more bZIP domain polypeptides or polypeptide subsequences.

The invention also provides an isolated nucleic acid or complementthereof, encoding a B3 domain polypeptide comprising a sequence at least50% identical to the B3 domain of SEQ ID NO: 2 or 4. In a preferredembodiment, the B3 domain comprises a polypeptide sequence from aboutamino acid 187 to about amino acid 295 of SEQ ID NO:4. In anotherembodiment, the B3 domain polypeptide comprises a polypeptide sequenceat least 50% identical to the B3 domain of SEQ ID NO:2 Such B3 domainpolypeptides can be encoded by polynucleotide sequences at least 39%identical to B3 domain sequences shown in SEQ ID NO: 1 (nucleotideposition 1560 to 1929) or SEQ ID NO:3 (nucleotide position 559-885),respectively. In another embodiment, the B3 domain polypeptide is afusion between two or more B3 domain polypeptides or polypeptidesubsequences.

The invention also provides an isolated nucleic acid or complementthereof, encoding a bZIP domain polypeptide comprising a subsequence atleast 60% identical to the bZIP domain of SEQ ID NO: 6. In a preferredembodiment, the bZIP domain comprises a polypeptide sequence betweenabout amino acid residue 353 and amino acid residue 417 of SEQ ID NO:6.Such bZIP domain polypeptides can be encoded by the polynucleotide atleast 39% identical to sequences shown in SEQ ID NO:5 (nucleotideposition 1057 to 1251). In another embodiment, the bZIP domainpolypeptide is a fusion between two or more bZIP domain polypeptides orpolypeptide subsequences.

In one embodiment, it may be advantageous for propagating or expressingthe polynucleotide to carry it in a bacterial or fungal strain with theappropriate vector suitable for the cell type. Common methods ofpropagating polynucleotides and producing proteins in these cell typesare known in the art and are described, for example, in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1982) and Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989).

The invention also provides isolated polypeptides comprising amino acidsequences at least 50% identical to the B3 domain of SEQ ID NO:2 or 4and capable of exhibiting at least one of the biological activities ofthe polypeptides encoded in SEQ ID NO:2, SEQ ID NO:4 or a fragmentthereof. Antibodies capable of binding the above described polypeptidesare also provided.

The invention also provides isolated polypeptides comprising amino acidsequences at least 60% identical to the bZIP domain of SEQ ID NO:6 andcapable of exhibiting at least one of the biological activities of thepolypeptides encoded in SEQ ID NO:6 or a fragment thereof. Antibodiescapable of binding the above described polypeptides are also provided.The invention also provides transgenic plant cells or plants comprisingan expression cassette comprising a promoter operably linked to aheterologous polynucleotide sequence, or complement thereof, encoding aB3 domain polypeptide comprising a sequence which is at least 50%identical to the B3 domain of SEQ ID NO:2 or 4. Such B3 domainpolypeptides can be encoded by the polynucleotide sequences shown in SEQID NO:1 or SEQ ID NO:3 respectively. The invention also provides plantsthat are regenerated from the plant cells discussed above.

The invention also provides transgenic plant cells or plants comprisingan expression cassette comprising a promoter operably linked to aheterologous polynucleotide sequence, or complement thereof, encoding abZIP domain polypeptide comprising a sequence which is at least 60%identical to the bZIP domain of SEQ ID NO:6. Such bZIP domainpolypeptides can be encoded by the polynucleotide sequences shown in SEQID NO:5. The invention also provides plants that are regenerated fromthe plant cells discussed above.

In activating transcription of a nucleic acid coding sequence, thetranscription factors described herein may interact with (1) a nativepromoter or (2) a non-native, recombinant or heterologous promoter. Ineither case, all or part of the promoter sequence is operably linked toa native nucleic acid coding sequence or a heterologous nucleic acidcoding sequence (e.g., a transgene) and may be from the same or adifferent species from that of the plant in which it is present. Thetransgene may be a reporter gene, such as luciferase (LUC) orβ-glucuronidase (GUS), or a gene encoding a recombinant protein that isexpressed in the plant.

In practicing the invention, a plant cell may be transformed with one ormore vectors, each comprising the coding sequence for one or more planttranscription factors, each operably linked to a tissue specificpromoter, wherein the tissue specific promoters may be the same ordifferent. It will be understood by those of skill in the art that onceexpressed a recombinant transcription factor may act on the promoterwhich is regulating expression of the transcription factor itself, oneor more heterologous target promoters, in addition to acting on multiplenative promoters.

In the case where the inserted polynucleotide sequence is transcribedand translated to produce a functional polypeptide, one of skill willrecognize that because of codon degeneracy a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the term “polynucleotide sequence from” aparticular B3 and/or bZIP domain gene. In addition, the termspecifically includes sequences (e.g., full length sequences)substantially identical (determined as described below) with a B3 and/orbZIP domain gene sequence and that encode proteins that retain thefunction of a B3 and/or bZIP domain polypeptide.

In the case of polynucleotides used to inhibit expression of anendogenous or heterologous gene, the introduced sequence need not beperfectly identical to a sequence of the target gene. The introducedpolynucleotide sequence will typically be at least substantiallyidentical (as determined below) to the target sequence.

Polynucleotide sequences according to the invention are suitable ashybridization probes for RNA, cDNA and DNA, in order to isolate thosecDNAs or genes which exhibit a high degree of similarity to the sequenceof the B3 and/or bZIP domain gene.

Polynucleotide sequences according to the invention are also suitable asprimers for polymerase chain reaction (PCR) for the production of DNAwhich encodes a protein having activity of a B3 and/or bZIP domainpolypeptide.

Oligonucleotides such as these, which serve as probes or primers, cancontain more than 30, preferably up to 30, more preferably up to 20,most preferably at least 15 successive nucleotides. Oligonucleotideswith a length of at least 40 or 50 nucleotides are also suitable.

The polypeptides according to the invention include polypeptidescorresponding to SEQ ID NO. 2 or 4, particularly those with thebiological activity of a B3 domain protein, and also includes those, atleast 50% of which, preferably at least 60% of which, are homologouswith the B3 domain of the polypeptides corresponding to SEQ ID NO. 2 and4 and which have the cited activity.

The polypeptides according to the invention include polypeptidescorresponding to SEQ ID No. 6, particularly those with the biologicalactivity of a bZIP domain protein, and also includes those, at least 60%of which are homologous with the bZIP domain of the polypeptidecorresponding to SEQ ID NO. 6 and which have the cited activity.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other, or a third nucleic acid,under stringent conditions. In the present invention, mRNA encoded by B3and/or bZIP domain genes of the invention can be identified in RNA blotsunder stringent conditions using cDNAs of the invention or fragments ofat least about 100 nucleotides. Genomic DNA or cDNA comprising genes ofthe invention can be identified using the same cDNAs specified in SEQ IDNO 1, 3, or 5 (or fragments of at least about 100 nucleotides) under lowstringency conditions for heterologous probing of samples.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):Tm=81.5° C.+16.6(log M)+0.41(% GC)-0.61 (% form)-500/L; where M is: themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with approximately 90% identity are sought, the Tm can bedecreased 10° C. Generally, stringent conditions are selected to beabout 5° C. lower than the thermal melting point (Tm) for the specificsequence and its complement at a defined ionic strength and pH. However,severely stringent conditions can utilize a hybridization and/or wash at1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (Tm); low stringencyconditions can utilize a hybridization and/or wash at 11, 12, 13, 14,15, or 20° C. lower than the thermal melting point (Tm). Using theequation, hybridization and wash compositions, and desired Tm, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a Tm of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inCurrent Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (2000). Thus,with the foregoing information, the skilled artisan can identify andisolate polynucleotides which are substantially similar to the presentpolynucleotides. In so isolating such a polynucleotide, thepolynucleotide can be used as the present polynucleotide in, forexample, increasing the stress tolerance of a plant.

The invention also relates to coding DNA sequences which result from SEQID NO. 1, SEQ ID NO: 3 and SEQ ID NO: 5 by degeneration of the geneticcode. In the same manner, the invention further relates to DNA sequenceswhich hybridize with SEQ ID NO. 1, 3, or 5 or with parts of SEQ ID NOs.1, 3, or 5. Moreover, one skilled in the art is also aware ofconservative amino acid replacements such as the replacement of glycineby alanine or of aspartic acid by glutamic acid in proteins as “sensemutations” which do not result in any fundamental change in the activityof the protein, i.e. which are functionally neutral. It is also knownthat changes at the N- and/or C-terminus of a protein do notsubstantially impair the function thereof, and may even stabilise saidfunction.

In the same manner, the present invention also relates to DNA sequenceswhich hybridize with SEQ ID NO. 1, 3, or 5 or with parts of SEQ ID NOs.1, 3, or 5. Finally, the present invention relates to DNA sequenceswhich are produced by polymerase chain reaction (PCR) usingoligonucleotide primers which result from SEQ ID NOs. 1, 3, or 5.Oligonucleotides of this type typically have a length of at least 15nucleotides.

In one preferred embodiment, the plant transcription factor is operablylinked to a tissue-specific promoter, preferably an inducible promoter.

In a preferred embodiment the polynucleotides of the present inventionare in a vector and/or a host cell. Preferably, the polynucleotides arein a plant cell or transgenic plant. In a preferred embodiment,transgenic plant lines, e.g., rice, wheat, corn, barley, oat, rape,cotton, peanut, and soybean are developed and genetic crosses carriedout using conventional plant breeding techniques. In one exemplaryapproach, a first stable transgenic plant line is generated where theplants express two transcription factors, e.g. 1) a B3-domaintranscription factor and a bZIP domain transcription factors, or 2) twodifferent B3-domain transcription factors, under the control of atissue-specific promoter. A number of such lines may be generated withvarying levels of transcription factor expression. In practicing themethod, these plants may be crossed with a parental transgenic rice,wheat, corn, barley, oat, rape, cotton, peanut, or soybean line thatexpresses a heterologous protein coding sequence (e.g., a recombinantprotein) under the control of a tissue-specific promoter that isresponsive to the transcription factors expressed in the first plantline. Plants derived from the resulting cross (F2) have a higherexpression level of the heterologous protein in one or more particularseed tissues, than a corresponding non-transgenic plant. In a preferredembodiment, the B3 domain transcription factor(s) and/or bZIP domaintranscription factor may be incorporated into the same (original) plantor into two separate transcription factor-expressing lines that arecrossed together to make a double transcription factor line wherein thetranscription factors will synergize with each other to control theexpression of endogenous genes and/or a third recombinant “target”. The“target” can also be crossed in and selected for “triple-expressing”lines.

In another preferred embodiment, the present invention provides methodsof increasing the stress tolerance of a plant in need thereof,comprising introducing the polynucleotides of the invention into saidplant.

In another preferred embodiment, the present invention provides anisolated polypeptide comprising the amino acid sequence in SEQ ID NO:2or 4 or 6 or those proteins that are at least 40%, preferably 50%,preferably 60% and preferably 95% identity to SEQ ID NO:2, 4, or 6.Preferably, the polypeptides have B3 domain or bZIP activity to effectexpression driven by an ABA-inducible promoter.

In another preferred embodiment, the present invention provides a methodfor making B3 or bZIP domain proteins, comprising culturing the hostcell carrying the polynucleotides of the invention for a time and underconditions suitable for expression of the B3 or bZIP, and collecting theB3 or bZIP protein.

In another preferred embodiment the present invention provides a processfor screening for polynucleotides which encode a B3 domain and bZIPdomain proteins comprising hybridizing the polynucleotide of theinvention to the polynucleotide to be screened; expressing thepolynucleotide to produce a protein; and detecting the presence orabsence of B3 domain activity in said protein.

One embodiment of the present invention is methods of screening forpolynucleotides in a sample which have substantial homology to thepolynucleotides of the present invention, preferably thosepolynucleotides encoding a protein having B3 domain activity. The methodcomprises providing an isolated B3 domain nucleic acid moleculecomprising a polynucleotide sequence, or complement thereof, encoding aB3 domain polypeptide with a subsequence at least 50% identical to theB3 domain of SEQ ID NO:2 or 4, contacting the isolated nucleic acidmolecule with a sample under conditions which permit a comparison of thesequence of the isolated nucleic acid molecule with the sequence of DNAin the sample; and analyzing the result of the comparison. In someembodiments, the isolated nucleic acid molecule and the sample arecontacted under conditions which permit the formation of a duplexbetween complementary nucleic acid sequences.

One embodiment of the present invention is methods of screening forpolynucleotides in a sample which have substantial homology to thepolynucleotides of the present invention, preferably thosepolynucleotides encoding a protein having bZIP domain activity. Themethod comprises providing an isolated bZIP domain nucleic acid moleculecomprising a polynucleotide sequence, or complement thereof, encoding abZIP domain polypeptide with a subsequence at least 60% identical to thebZIP domain of SEQ ID NO:6, contacting the isolated nucleic acidmolecule with a sample under conditions which permit a comparison of thesequence of the isolated nucleic acid molecule with the sequence of DNAin the sample; and analyzing the result of the comparison. In someembodiments, the isolated nucleic acid molecule and the sample arecontacted under conditions which permit the formation of a duplexbetween complementary nucleic acid sequences.

In a preferred embodiment the B3 domain polypeptide and the bZIP domainpolypeptide have functional assays for B3 or bZIP transacting activityon ABA-inducible gene expression.

In another preferred embodiment, the present invention includes aprocess for screening in a transient functional assay forpolynucleotides which encode a protein having B3 or bZIP transactingactivity on ABA-inducible gene expression comprising hybridizing thepolynucleotides of the invention to the polynucleotide to be screened;expressing the polynucleotide to produce a protein; and detecting thepresence or absence of B3 or bZIP trans-acting activity on ABA-induciblegene expression in said functional assay. In another preferredembodiment, the present invention provides a method for making B3 orbZIP proteins, comprising culturing the host cell carrying thepolynucleotides of the invention for a time and under conditionssuitable for expression of B3 or bZIP proteins, and collecting the B3 orbZIP proteins protein.

In the case of both expression of transgenes and inhibition ofendogenous genes (e.g., by antisense, or sense suppression) one of skillwill recognize that the inserted polynucleotide sequence need not beidentical and may be “substantially identical” to a sequence of the genefrom which it was derived. As explained below, these variants arespecifically covered by this term.

In another preferred embodiment, the present invention provides a methodfor detecting a nucleic acid with at least 48% homology to the B3 domainnucleotide SEQ ID NO:1 or 3 or 5, sequences which are complimentary toSEQ ID NO:1 or 3 or 5 and/or which encode a protein having substantialidentity with the amino acid sequence in SEQ ID NO:2, 4 or 6respectively, comprising contacting a nucleic acid sample with a probeor primer comprising at least 15 consecutive nucleotides of thenucleotide sequences of SEQ ID NO:1, 3, or 5 or at least 15 consecutivenucleotides of the complement thereof.

Thus, in one embodiment of the present invention, the stress toleranceof a plant can be enhanced or increased by increasing the amount ofprotein available in the plant, preferably by the enhancement of the B3and/or bZIP domain gene expression in the plant. Thus, one embodiment ofthe present invention are plant cells carrying the polynucleotides ofthe present invention, and preferably transgenic plants carrying theisolated polynucleotides of the present invention.

Plant Transcription Factors

Transcription factors are capable of sequence-specific interaction witha gene sequence or gene regulatory sequence. The interaction may bedirect sequence-specific binding in that the transcription factordirectly contacts the gene or gene regulatory sequence, or indirectsequence-specific binding mediated by interaction of the transcriptionfactor with other proteins. In these cases, the binding and/or effect ofthe B3-domain transcription factor is influenced in a synergistic mannerby another B3-domain transcription factor or a bZIP-domain transcriptionfactor.

The gene or gene regulatory region and transcription factor may bederived from the same type of plant (e.g., the same species or genus) ora different type of plant. The transcription factors used herein producesynergistic results related to the ability of the plant to respond tostress. The B3-domain transcriptions factors and the bZIP transcriptionsfactors are described in detail above.

Constructs for Expression of a Transcription Factor in a Plant Cell

A heterologous nucleic acid construct or expression vector designed foroperation in plants comprising the coding sequence for a planttranscription factor may be used to transiently or stably transform aplant, e.g. a monocot plant. An exemplary heterologous nucleic acidconstruct or expression vector designed for operation in plants,includes (i) a promoter (transcriptional regulatory region) induced inparticular tissue (“tissue-specific”), (ii) the coding sequence for aplant transcription factor operably linked to the promoter, (iii)companion sequences upstream and downstream which are of plasmid orviral origin and provide necessary characteristics to the vector topermit the vector to move the DNA from bacteria to the desired planthost; (iv) a selectable marker sequence; and (v) a transcriptionaltermination region generally at the opposite end of the vector from thetranscription initiation regulatory region. Suitable transformationvectors for the preparation of such constructs are known in the art andmany are commercially available.

Vector components may also include a signal sequence. The desiredrecombinant protein or polypeptide may be produced directly, or as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. Included inheterologous nucleic acid constructs for use in the methods of theinvention are signal sequences that allow processing and translocationof the protein, as appropriate.

In some cases, the recombinant protein may be produced as a precursorprotein, which may be further processed in the plant cell culture orfollowing extraction from the plant.

Tissue-Specific Promoters

The transcription regulatory or promoter region of the chimeric gene orheterologous nucleic acid construct is preferably a tissue-specificpromoter, for example, a promoter capable of directing expression of agene product under its control, which is specific to the seed embryo,aleurone, outer layer of the endosperm, mesophyll cells, vascular cells,guard cells, and the like.

Promoter sequences for regulating transcription of operably linkedcoding sequences include naturally-occurring promoters, or regionsthereof capable of directing tissue-specific transcription, and hybridpromoters, which combine elements of more than one promoter. Methods forconstruction of hybrid promoters are well known in the art. In somecases, the promoter is derived from the same plant species as the plantin which the nucleic acid construct is to be introduced. Promoters foruse in the invention are typically derived from crops such as rice,barley, wheat, corn, sunflower, carrot, bean, rape, and model speciessuch as Arabidopsis.

Alternatively, a tissue-specific promoter from one type of monocot maybe used to regulate transcription of a gene coding sequence from adifferent monocot or dicot. Numerous types of appropriate expressionvectors, and suitable regulatory sequences are known in the art for avariety of plant host cells. In general, the transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences.

Effective inducible or tissue-specific transcriptional initiationregions, e.g., promoters, may be isolated from various tissues and/or atvarious stages of development by a variety of techniques routinely usedby those of skill in the art, including, but not limited to: (1)conventional hybridization techniques using known coding sequences froma different species, tissue and/or developmental stage, followed bycharacterizing the region 5′ of the homologous gene to identify theassociated transcriptional initiation sequence; (2) subtractivehybridization, (3) differential display, and (4) selective amplificationvia biotin- and restriction-mediated enrichment, SABRE.

Expression Vector Components

Expression vectors or heterologous nucleic acid constructs, designed foroperation in plants, comprise companion sequences upstream anddownstream from the expression cassette. The companion sequences are ofplasmid or viral origin and provide necessary characteristics to thevector to permit the vector to move DNA from bacteria to the plant host,such as, sequences containing an origin of replication and a selectablemarker. Typical secondary hosts include bacteria and yeast.

The transcriptional termination region may be taken from a gene where itis normally associated with the transcriptional initiation region or maybe taken from a different gene.

The particular marker gene employed is one that allows for selection oftransformed cells as compared to cells lacking the DNA that has beenintroduced. Preferably, the selectable marker gene is one thatfacilitates selection at the tissue culture or seedling stages.

In general, a selected nucleic acid sequence is inserted into anappropriate restriction endonuclease site or sites in the vector.Standard methods for cutting, ligating and E. coli transformation, knownto those of skill in the art, are used in constructing vectors for usein the present invention. Generally, vectors for use in practicing thepresent invention are constructed using methods known to those skilledin the art.

Plants

The plants used in practicing the invention are of both monocot anddicot origin. The Graminaceae family includes all members of the grassfamily of which the edible varieties are known as cereals or grains. Thecereals include a wide variety of species such as wheat (Triticum sps.),rice (Oryza sps.), barley (Hordeum sps.), oats (Avena sps.), rye (Secalesps.), corn (Zea sps.), and millet (Pennisettum sps.). In one embodimentof the invention, preferred family members are rice, wheat, corn,barley, oat, rape, cotton, peanut, and soybean.

Plant cells or tissues derived from the members of the family may betransformed with expression vectors (i.e., plasmid DNA into which thegene of interest has been inserted) using a variety of standardtechniques (e.g., microparticle bombardment, electroporation, protoplastfusion or infection with Agrobacterium).

Transgenic plant cells obtained as a result of such transformationexpress the coding sequence for one or more plant transcription factor,e.g. a B3-domain transcription factor and bZIP or B3-domaintranscription factors. The transgenic plant cells are cultured in mediumcontaining the appropriate selection agent to identify and select forplant cells which express the heterologous nucleic acid sequence. Afterplant cells that express the heterologous nucleic acid sequence areselected, whole plants are regenerated from the selected transgenicplant cells. Techniques for regenerating whole plants from transformedplant cells are generally known in the art.

In one embodiment of the invention, transgenic plant lines, e.g., rice,wheat, corn, barley, oat, rape, cotton, peanut, and soybean aredeveloped and genetic crosses carried out using conventional plantbreeding techniques. In one example of this approach, a first stabletransgenic plant line is generated where the plants express two of thetranscription factors under the control of a tissue-specific promoter. Anumber of such lines may be generated with varying levels oftranscription factor expression. The plants are crossed with a secondtransgenic plant line that expresses a heterologous protein codingsequence (e.g., a recombinant protein) under the control of atissue-specific promoter that is responsive to the transcription factorsexpressed in the first plant line. The resulting cross (F2) has a higherexpression level of the heterologous protein in one or more particulartissues, dependent upon the promoter used.

Transformation of Plant Cells

Vectors useful in the practice of the present invention may bemicroinjected directly into plant cells by use of micropipettes tomechanically transfer the nucleic acid construct or cassette. Suchnucleic acid constructs or cassettes may also be transferred into theplant cell using polyethylene glycol. In addition, high velocityballistic penetration by small particles with the nucleic acid eitherwithin the matrix of small beads or particles, or on the surface mayalso be used for introduction of nucleic acid sequences into plantcells.

Additional methods for introduction of nucleic acid sequences into plantcells include fusion of protoplasts with other entities, eitherminicells, cells, lysosomes or other fusible forms for introduction ofnucleic acid sequences into plant cells with lipid surfaces; andelectroporation. In this technique, electrical impulses of high fieldstrength reversibly permeabilize biomembranes allowing the introductionof plasmids into plant cells or protoplasts. Electroporated plantprotoplasts will reform the cell wall, divide, and form plant callus.

Another preferred method of introducing a nucleic acid construct intoplant cells is to infect a plant cell, explant, meristem or seed withAgrobacterium, in particular Agrobacterium tumefaciens. A nucleic acidconstruct comprising such a sequence of interest can be introduced intoappropriate plant cells.

Standard Agrobacterium binary vectors are known to those of skill in theart and many are commercially available. Expression vectors typicallyinclude polyadenylation sites, translation regulatory sequences (e.g.,translation start sites), introns and splice sites, enhancer sequences(which can be inducible, tissue-specific or constitutive), and mayfurther include 5′ and 3′ regulatory and flanking sequences.

Suitable selectable markers for selection in plant cells are describedabove and the particular marker gene employed is one which allows forselection of transformed cells as compared to cells lacking the DNAwhich has been introduced. Preferably, the selectable marker gene is onewhich facilitates selection at the tissue culture or seedling stages.

Transformed explant cells are screened for the ability to be cultured inselective media having a threshold concentration of selective agent.Explants that can grow on the selective media are typically transferredto a fresh supply of the same media and cultured again. The explants arethen cultured under regeneration conditions to produce regenerated plantshoots. After shoots form, the shoots are transferred to a selectiverooting medium to provide a complete plantlet. The plantlet may then begrown to provide seed, cuttings, or the like for propagating thetransformed plants. The method provides for efficient transformation ofplant cells with expression of modified native or non-native plant genesand regeneration of transgenic plants, which can produce a recombinantprotein or polypeptide of interest.

The expression of a recombinant protein or polypeptide can be confirmedusing any of a number of standard analytical techniques such as Westernblot, ELISA, PCR, HPLC, NMR, or mass spectroscopy.

PREFERRED EMBODIMENT

Strong and novel activities in rice embryonic and maize mesophyllprotoplasts of the transcription factors VP1 (see SEQ. ID. NO: 1 andSEQ. ID. NO.:2), RAV2 (see SEQ. ID. NO.:3 and SEQ. ID. NO.:4), ABI5 (seeSEQ. ID. NO.:5 and SEQ.ID.NO.: 6), ABF1, ABF3, ABF4, AREB3 and DPBF4genes from maize (VP1) and Arabidopsis (all others) are demonstratedherein. As shown in the Examples below, the ABA INSENSITIVE-5 (ABI5)basic leucine zipper (bZIP) domain transcription factor and closelyrelated ABI-5-like homologues have been tested for transactivation ofthe ABA-inducible wheat Em promoter in transiently transformed rice andmaize protoplasts. The functional interactions of co-expressed ABI5 andthe ABI5-like ABA-Response Element-Binding Factors, such as ABF1, ABF2,ABF3, ABF4, AREB3, and DPBF4/EEL, which have highly conserved domains(C1-C3, bZIP), were tested with each other and with co-expressed maizeVIVIPAROUS-1 (VP1) B3-domain transcription factor. OverexpressedArabidopsis ABI5, ABF3, ABF4, AREB3, DPBF4, and maize VP1, but not ABF1and ABF2, individually show synergy with ABA in rice embryonic and/ormaize mesophyll protoplasts. However, when ABI5 and ABF3 areco-expressed in rice protoplasts, they show no synergistic interactionswith each other, in contrast to strong synergy observed with ABI5, orABF3 co-expressed with VP1 in rice or maize. Furthermore, AREB3,DPBF4/EEL, and ABF4 do not work in synergy with co-transformed maizeVP1, whereas ABI5, ABF3 and ABF1 can synergize with VP1 in maizemesophyll protoplasts. This latter result is in contrast to theobservation that ABF1 does not appear to interact with ABA alone. Takentogether, these functional data provide several examples to support theclaim that formulations of bZIP domain transcription factors withB3-domain transcription factors will provide novel and useful means toeffect stress- and ABA-inducible gene expression that in turn willenhance valuable traits such as productivity, yields, and stresstolerance of commercial varieties of plants. The data provides strongevidence that expression of these and homologous transgenes from, andinto, various plant species in target tissues such as leaves or seedswill render transgenic horticultural and ornamental plants and crops tobe better able to withstand environmental stress via coordinatedregulation of multiple endogenous gene sets in stress tolerancepathways.

In the Examples and previously published results, it has beendemonstrated that over-expression of maize VP1, Arabidopsis ABI5, orseveral ABI5-related family members transactivate various ABA-induciblepromoters from both monocots and dicots in rice or maize protoplasts,proving that these transcription factors are key targets of a conservedABA signaling pathway in plants. Others have shown that ectopicexpression of ABI3, ABI4, or ABI5 transcription factors results in ABAhypersensitivity of vegetative tissues which is partly dependent onincreased ABI5 expression. Taken together, these results show that thesetranscription factors participate in combinatorial control of geneexpression, possibly by forming a regulatory complex mediatingseed-specific and/or ABA-inducible expression.

Table 1 documents the polypeptide and nucleotide similarities of thebZIP domains of select Arabidopsis genes compared to ABI5. Significantfunctional effects have been shown on transactivation of the Em promoterfor most of these family members. The percentage identity and similarityresults for polypeptides are relative, since the BLASTP alignmentalgorithm does not necessarily take into account all residues of thebZIP. The E values are directly comparable measures of significanthomologies between and among pairs of genes. (Altschul et al., GappedBLAST and PSI-BLAST: a new generation of protein database searchprograms. Nucleic Acids Res. 25:3389-3402 (1997)). For example, thedifference in E values between ABF3.2 and ABF3.1 is 1000-fold relativelyhigher homology of ABF3.2 to ABI5 than ABF3.1.

TABLE 1 Pairwise comparison of polypeptide and nucleotide homologiesbetween ABI5-Like Family members and ABI5 bZIP domains. Percent Percent% n.t. Identity with SEQ. ID. NO. Identity of ABI5bZIP Similarity ABI5bZIP (n.t. 1057-1251, 1) nucleotides AGI Annotation (aa 353-417, SEQ.NO. 6) ABI5 bZIP E SEQ NO. 5) 2) protein At2g36270 ABI5 100 — 4.E−30100 1) SEQ. ID. NO. 5 2) SEQ.ID.NO.: 6 At1g49720 ABF1 62 72 1.E−14 52 1)SEQ.ID.NO.: 7 2) SEQ.ID.NO.: 8 At5g42910 ABF2-Like 50 69 6.E−10 53 1)SEQ.ID.NO.: 9 2) SEQ.ID.NO.: 10 At1g45249 ABF2 71 81 3.E−17 62 1)SEQ.ID.NO.: 11 2) SEQ.ID.NO.: 12 At4g34000 ABF3.2 66 80 8.E−18 56 1) SEQID. NO.: 13 2) SEQ.ID.NO.: 14 At4g34000 ABF3.1 80 93 8.E−15 52 1)SEQ.ID.NO.: 15 2) SEQ ID. NO.: 16 At3g19290 ABF4 63 80 1.E−16 58 1)SEQ.ID.NO.: 17 2) SEQ.ID.NO.: 18 At3g56850 AREB3 61 73 3.E−13 39 1)SEQ.ID.NO.: 19 2) SEQ.ID.NO.: 20 At3g44460 DPBF2 67 80 6.E−18 52 1)SEQ.ID.NO.: 21 2) SEQ.ID.NO.: 22 At2g41070 DPBF4/EEL 69 87 1.E−13 48 1)SEQ.ID.NO.: 23 2) SEQ.ID.NO.: 24 At1g03970 GBF4 60 72 4.E−12 48 1)SEQ.ID.NO.: 25 2) SEQ.ID.NO.: 26 At5g44080 GBF4-Like 63 73 1.E−12 54 1)SEQ.ID.NO.: 27 2) SEQ.ID.NO.: 28

Table 2 documents that the VP1 B3-domain protein sequence can be used toclaim Arabidopsis ABI3, LEC2, FUS3, and At4g21550(=AB3L3), whereas theRAV2 protein and/or its B3 domain can be used to claim “more preferredembodiments” (>50% identity or similarity) with At1g13260=RAV1,At3g25730=RAV1-Like, At1g25560=RAV2-Like, 2g30470=B3L1, At4g32010=AB3L2,At4g01500=AB3L4, At1g51120=AB3L5, At2g46870=AB3L6, At1g01030=AB3L7,At2g36080=AB3L8, At 1 g50680=AB3L9, At3g61970=AB3L10, At5g06250=AB3L11,At3g11580=AB3L12, and At2g28350=AB3L16.

The AGI indications in both Table 1 and Table 2 can be used to obtainadditional information about the specific proteins and polynucleotideson the website www.arabidopsis.org.

TABLE 2 Pairwise homologies between Arabidopsis ABI3-Like family membersand the VIVIPAROUS1 B3 domain or RAV2 protein.* Percent Identity withVP1b3 Percent Identity with RAV2 AGI number Annotation (aa496-619 SEQ IDNO. 2) Percent Similar VP1b3 E SEQ. ID NO. 4) Percent Similar with RAV2At3g24650 ABI3 84 90 2.E−56 At3g26790 FUS3 57 74 7.E−32 At1g28300 LEC252 70 1.E−29 At1g13260 RAV1 36 54 2.E−09 67 78 At3g25730 RAV1- 35 512.E−09 62 75 Like At1g68840 RAV2 33 46 6.E−11 100 — At1g25560 RAV2- 3550 9.E−10 75 83 Like At2g30470 AB3L1 42 60 2.E−17 43 65 at4g32010 AB3L240 60 6.E−17 40 65 At4g21550 AB3L3 38 62 6.E−14 At4g01500 AB3L4 31 512.E−09 60 73 At1g51120 AB3L5 34 51 2.E−08 39 58 At2g46870 AB3L6 32 522.E−09 65 80 At1g01030 AB3L7 32 55 9.E−08 54 70 At2g36080 AB3L8 31 484.E−07 66 79 At1g50680 AB3L9 32 47 3.E−07 39 56 At3g61970 AB3L10 31 491.E−07 67 83 At5g06250 AB3L11 29 45 3.E−07 63 77 At3g11580 AB3L12 28 433.E−07 57 71 At2g28350 AB3L16  29§  38§ 0.42 35 53 % n.t. identity withRAV2 B3 SEQ.ID. NO: Percent Identity with RAC2b3 domain (nt. 559-885 SEQID 1) nucleotides AGI number E 87-295, SEQ ID NO. 4) Percent Similarwith RAV2b3 E NO. 3) 2) protein At3g24650 34 49 3.E−10 48 1) SEQ.ID.NO.:29 2) SEQ.ID.NO.: 30 At3g26790 39 58 1.E−10 47 1) SEQ.ID.NO.: 31 2)SEQ.ID.NO.: 32 At1g28300 34 50 1.E−09 49 1) SEQ.ID.NO.: 33 2)SEQ.ID.NO.: 34 At1g13260 1.E−111 86 92 3.E−50 65 1) SEQ.ID.NO.: 35 2)SEQ.ID.NO.: 36 At3g25730 1.E−101 73 85 6.E−43 62 1) SEQ.ID.NO.: 37 2)SEQ.ID.NO.: 38 At1g68840 0.E+00 100 — 4.E−61 100 1) SEQ.ID.NO.: 3 2)SEQ.ID.NO.: 4 At1g25560 1.E−135 83 86 2.E−50 66 1) SEQ.ID.NO.: 39 2)SEQ.ID.NO.: 40 At2g30470 1.E−13 43 65 1.E−14 61 1) SEQ.ID.NO.: 41 2)SEQ.ID.NO.: 42 at4g32010 5.E−13 40 65 5.E−14 50 1) SEQ.ID.NO.: 43 2)SEQ.ID.NO.: 44 At4g21550 40 59 4.E−11 56 1) SEQ.ID.NO.: 45 2)SEQ.ID.NO.: 46 At4g01500 2.E−33 59 73 1.E−33 74 1) SEQ.ID.NO.: 47 2)SEQ.ID.NO.: 48 At1g51120 3.E−39 51 67 6.E−22 68 1) SEQ.ID.NO.: 49 2)SEQ.ID.NO.: 50 At2g46870 4.E−39 68 83 6.E−39 78 1) SEQ.ID.NO.: 51 2)SEQ.ID.NO.: 52 At1g01030 3.E−40 69 82 3.E−39 59 1) SEQ.ID.NO.: 53 2)SEQ.ID.NO.: 54 At2g36080 6.E−38 68 79 5.E−37 58 1) SEQ.ID.NO.: 55 2)SEQ.ID.NO.: 56 At1g50680 7.E−41 50 65 8.E−22 68 1) SEQ.ID.NO.: 57 2)SEQ.ID.NO.: 58 At3g61970 3.E−38 67 83 2.E−38 76 2) SEQ.ID.NO.: 59 1)SEQ.ID.NO.: 60 At5g06250 8.E−37 64 77 3.E−37 74 1) SEQ.ID.NO.: 61 2)SEQ.ID.NO.: 62 At3g11580 2.E−38 62 77 8.E−36 48 1) SEQ.ID.NO.: 63 2)SEQ.ID.NO.: 64 At2g28350 7.E−10 36 54 1.E−10 51 1) SEQ.ID.NO.: 65 2)SEQ.ID.NO.: 66 *Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z,Miller W, Lipmann DJ (1997) Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res 25: 3389-3402.See http://www.arabidopsis.org/ {circumflex over ( )} Higgins D,Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignmentthrough sequence weighting, position-specific gap penalties and weightmatrix choice. Nucleic Acids Res. 22: 4673-4680. See EuropeanBioinformatics Institute http://www.ebi.ac.uk/clustalw §Compared to ABI3B3 domain (aa551-674, SEQ ID NO. 30).

Example 1 Materials and Methods

Plant Materials. Maize mesophyll protoplasts were isolated from 20-hrilluminated leaves of 10 day old maize seedlings that had been kept inthe dark at 25° C. The middle part of the second leaves (about 6 cm inlength) was cut into 0.5 mm strips with a razor blade and digested in anenzyme solution containing 1% (w/v) cellulose RS, 0.1% (w/v) macerozymeR10 (Yakult Honsha, Nishinomiya, Japan), 0.6 M mannitol, 10 mM MES (pH5.7), 1 mM CaCl₂, 1 mM MgCl₂, 10 mM β-mercaptoethanol, and 0.1% BSA(w/v) for 3 hr at room temperature. Protoplasts were released by shakingon a rotary shaker at 80 rpm for 10 min and were filtered through a 70μm nylon filter. Protoplasts were collected by centrifugation at 100 gfor 2 min, washed in cold 0.6 M mannitol solution, centrifuged, andresuspended at 2×10⁶/mL in cold 0.6 M mannitol. Electroporationconditions were 400 V/cm, 200 μF, 10 msec, and two pulses with a BioradGenePulser apparatus. Each sample contained 5×10⁴ protoplasts and about50 μg DNA in 0.3 mL of 0.6 M mannitol and 20 mM KCl.

Embryonic rice (Oryza sativa) callus cultures (Radon 6 from theInternational Rice Research Institute, Los Bafios, Phillipines) wereobtained. Embryonic rice callus cultures were grown as suspensions inliquid culture as well as on phytagel plates containing MS mediumsupplemented with 2.0 mg/L 2,4-D. Cultures were propagated and digestedfor making protoplasts as previously described except that 10 mM HEPES(Sigma, St. Louis, Mo., USA), pH 5.6, was substituted for phosphate inthe Krens'F medium, and 2% (weight/volume; w/v) cellulase YC, 0.35%(w/v) macerozyme, and 0.1% (w/v) pectolyase Y23 were used for overnightdigestion (Karlan Research Products, Santa Rosa, CA, USA). Protoplastswere transformed with various mixtures of DNA reporter and effectorconstructs using polyethylene glycol precipitation. Transformedprotoplasts were incubated with or without 100 μM ABA for 16 h in thedark in Krens solution before quantifying β-glucuronidase (GUS) andluciferase (LUC) reporter enzyme activities as previously described. ABAwas dissolved and stored in absolute ethanol at −20° C. as a 0.1 M stocksolution. Prior to use, required dilutions of ABA were made in Krenssolution, and control samples received the same volume of solvent as inABA treatments.

Plasmid Constructs

Plasmid pBM207 contains the wheat (Triticum aestivum) EarlyMethionine-labeled (Em) promoter driving the expression of GUS, encodedby uida from Escherichia coli. Plasmid pDH359 contains ABI5 cDNA drivenby Ubiquitin promoter. Plasmid pCR349.13S contains the 35S promoterdriving the VP1 sense cDNA. Plasmid pDirect2.6 contains the Ubi promoterin a reverse orientation and was used as control construct to balancethe total amount of input plasmid DNA between various treatments.Plasmid pAHC 18 contains the Ubi promoter driving firefly (Photinuspyralis) LUC cDNA and was included in transformations to provide aninternal reference for non-ABA-inducible transient transcription inreporter enzyme assays. ABF1-ABF4, AREB3, and DPBF4 were amplified byPCR using gene-specific primers from an Arabidopsis cDNA library (Minetet al. Plant J. 2:417 (1992)) and were cloned into plasmid pDH349(Gampala et al. J. Biol. Chem. 277: 1689 (2002)) containing the maizeUbiquitin promoter and nopaline synthase 3′ termination signals. Primersused for PCR amplification are listed in Table 3.

TABLE 3 Gene-specific PCR primers used to clone Arabidopsis ABI5-likecDNAs used herein. Primer sequence (5′->3′; Gene F = forward, R= reverse) ABF1 SEQ ID NO 67 F: cccaagcttggatccaaagggtctgattcgtttgt SEQID NO 68 R: cggggtaccgttaacgtcacatcttctctatagct ABF2 SEQ ID NO 69 F:cccaagcttggatcccccaaacgaagaaccaaaca SEQ ID NO 70 R:cggggtaccgatatcttcttcaaaattggtaactc ABF3 SEQ ID NO 71 F:ccgctcgagggatccgaagcttgatcctcctagtt SEQ ID NO 72 R:cggggtaccgatatcagatacaagataaattcact ABF4 SEQ ID NO 73 F:cccaagcttggatccgaacaagggttttagggctt SEQ ID NO 74 R:cggggtaccgatatcgttgccactcttaagtaata AREB3 SEQ ID NO 75 F:cccactagtggatccatggattctcagaggggtat SEQ ID NO 76 R:cggggtaccgatatctcagaaaggagccgagcttg DPBF4 SEQ ID NO 77 F:cccggtaccggatccacagtttctaaggcaaaata SEQ ID NO 78 R:cggaggcctgaattcacttgaactagtgtttgtac

Results

Previous results demonstrated that overexpressed ABF1 and ABF3 hadpositive effects on ABA-inducible Em-GUS reporter gene expression intransiently transformed rice protoplasts, providing functional evidencefor the involvement of these proteins in ABA- and stress signaltransduction. Simultaneously, it has been shown that ABF2, ABF3 and ABF4overexpression in transgenic Arabidopsis results in ABA hypersensitivityand other ABA-associated phenotypes such as altered ABA-inducible geneexpression and glucose signaling, reduced transpiration, and enhanceddrought tolerance. The functional roles of the ABI5-like family membersABF1-ABF4 in regulation of ABA-inducible gene expression in riceprotoplasts were tested and the results are shown in FIG. 1. FIG. 1Ashows that the overexpression of ABF3 and ABF4 is sufficient totransactivate ABA-inducible Em-GUS expression in rice protoplasts.Protoplasts were transformed with Em-GUS and a “dummy” effectorconstruct (pD2.6 containing only the Ubiquitin promoter used to driveeffector expression) or co-transformed with a Ubi-ABF construct andtreated with or without 100 μM ABA. The values shown in FIG. 1A are theaverage (±S.E.M.) of four replicate transformations. Consistent withprevious results, overexpressed ABF1 and ABF3 had slight and strongsynergy with exogenous ABA, respectively. However, overexpression ofABF2 had no effect on Em-GUS expression (see FIG. 1A). Interestingly,overexpression of ABF3 or ABF4 was sufficient for transactivation of theEm promoter. These results are consistent with those of previousresearchers who showed that overexpressed ABF3 or ABF4 resulted inaccumulation of the LEA genes rd29A and rab18.

FIG. 1B displays the results of an ABA-inducible reporter geneexpression experiment with transiently-transformed rice protoplastsoverexpressing ABI5, ABF3, and VP1 transcription factors alone and inpairwise co-transformations. FIG. 1B shows that the overexpressed ABF3interacts synergistically with ABA and VP1, but not with ABI5. Riceprotoplasts were transformed with either Em-GUS alone or in pairwisecombinations of Ubi-ABF3, Ubi-ABI5, or 35S-VP1. The values in FIG. 1Bare the average (±S.E.M.) of four replicate transformations. ABF3 is arelated member of the ABI5-family of bZIP transcription factors. Aspreviously reported, ABI5, ABF3, and VP1 transactivated the Em promoterand acted in synergy with ABA. More importantly, ABF3 and VP1 synergizedwith each other and with ABA when co-expressed. This activity was abouttwice the synergistic activity seen between ABI5 and VP1. However,paired expression of ABF3 and ABI5 bZIPs in protoplasts did not resultin synergy, see FIG. 1B. Based on these two examples of ABI5-familymember synergy with maize VP1, it is proposed that most members of theABI5 bZIP family can have functional interactions with VP1 and ABI3,including homologues from various species.

To provide evidence to support the claim that transcriptional regulationof ABA signaling is highly conserved among higher plants and indifferent tissue types, various ABI5-like homologues from Arabidopsiswere tested for their ABA signaling activities and functionalinteractions with VP1 in maize mesophyll protoplasts. The results areshown in FIG. 2. FIG. 2A shows that AREB3 or DPBF4/EEL overexpression inmaize mesophyll protoplasts is sufficient to transactivate Em-GUSexpression. Furthermore, ABF1 and ABF3, but not ABF2, AREB3, ABF4, orDPBF4/EEL, can synergize with co-transformed VP1 (FIG. 2B). The valuesin FIG. 2 are the average (±S.E.M.) of four replicate transformations.As previously shown for rice embryonic protoplasts (see FIG. 1), ABI5,ABF3 and ABF4 synergize with exogenous ABA and are sufficient fortransactivation of the Em promoter when overexpressed in maize mesophyllprotoplasts (FIG. 2A). ABF1 and ABF2 have lower levels of ABA synergycompared to ABF3 and ABF4 in maize (FIG. 2A), similar to activitiesobserved in rice (see FIG. 1). Furthermore, the ABI5-like family membersAREB3 and DPBF4/EEL synergize with exogenous ABA and are sufficient whenoverexpressed to transactivate the Em promoter (see FIG. 2A). Thisfinding demonstrates that ABI5-like family members can function in ABAsignaling and suggests they may have novel/unique functions andactivities including interactions with VP1 and VP1-like homologues.Indeed, ABF1 showed a strong synergy with VP1 in maize protoplastsdespite showing little synergy with ABA (compare FIG. 2B with FIG. 2A),unlike ABI5 and ABF3, which showed similar interactions with ABA and VP1as seen in rice, (compare FIG. 1 and FIG. 2). Interestingly, ABF4 andAREB3 showed no functional interactions with VP1 in maize mesophyllprotoplasts, while DPBF4/EEL showed some antagonism of ABA inducibleEm-GUS expression when co-expressed with VP1 (see FIG. 2B). This latterresult is consistent with the published genetic and biochemical evidencein Arabidopsis that DPBF4/EEL competes directly with ABI5 for functionalinteractions with ABI3 in transactivating ABA inducible promoters.Therefore, both similarities and differences are seen in the functionalinteractions of Arabidopsis ABI5-like genes with VP1. It is proposedthat other VP1-like family members of Arabidopsis, of which there areover 20, may be the cognate partners of ABI5-like family members such asABF2 and ABF4 that may regulate distinct, or tissue-specific aspects ofABA and stress signaling.

Example 2

To test the involvement of ABI3-Like B3 domain transcription factors inABA signaling, ABA-inducible reporter gene transactivation by the B3domain-containing RAV2 was assayed in maize mesophyll protoplaststransiently co-transformed with a full length cDNA under transcriptionalcontrol of the Ubiquitin promoter. Activation of Em-GUS reporter geneexpression by RAV2, relative to non-ABA inducible Ubi-LUC referencereporter as internal control, and functional interactions of RAV2 withVP1, ABI5, and the ABI5-Like ABF3 were compared in protoplasts treatedwith or without 100 uM ABA (see FIG. 3). The results in FIG. 3A showthat: 1) the novel B3 domain transcription factor RAV2, ABI5, and ABF3are sufficient for specific Em-GUS transactivation; and 2) RAV2synergizes with ABI5 and ABF3, similar to the results in FIGS. 1 and 2provided above. Furthermore, results shown in FIG. 3B demonstrate thatmaize VP1 and Arabidopsis RAV2, a ABI3-Like B3 domain transcriptionfactor, act synergistically to activate Em-GUS expression, demonstratingthat these novel interactions involve mechanisms conserved betweenmonocots and dicots. Furthermore, this synergism is enhanced by ABA, asshown in FIG. 3B. RAV2 transactivation is antagonized byco-transformation of the ABA-INSENSITIVE-1 dominant negative allele(data not shown), which support the notion that the RAV2 mechanismreported here is specific to ABA signaling.

Although the present invention has been disclosed in terms of apreferred embodiment, it will be understood that numerous additionalmodifications and variations could be made thereto without departingfrom the scope of the invention as defined by the following claims:

1. A method of producing a transgenic plant comprising a) transforming aplant cell with an expression vector comprising i) a B3-domaintranscription factor having a polynucleotide sequence with a B3 domainwhich is at least 25% identical to the B3 domain of SEQ. ID NO:1 or SEQ.ID. NO.: 3 and ii) a bZIP transcription factor having a polynucleotidesequence with a bZIP domain which is at least 25% identical to the bZIPdomain of SEQ. ID NO: 5, and b) generating from the plant cell atransgenic plant with an increased tolerance to environmental stress ascompared to a wild type variety of the plant.
 2. The method of claim 1,wherein said B3-domain transcription factor is VP1 as defined bySEQ.ID.NO.:
 1. 3. The method of claim 2, wherein said bZIP domaintranscription factor is ABI5 as defined by SEQ.ID.NO.: 5 or ABF1 asdefined by SEQ.ID.NO.:
 7. 4. The method of claim 1, wherein said bZIPdomain transcription factor is ABI5 as defined by SEQ.ID.NO.: 5 or ABF1as defined by SEQ.ID.NO.:
 7. 5. The method of claim 1, wherein said bZIPdomain transcription factor is selected from a group of polynucleotidesconsisting of SEQ.ID.NO. 9, SEQ.ID.NO. 11, SEQ.ID.NO. 15, SEQ.ID.NO. 17,SEQ.ID.NO.19, SEQ.ID.NO. 21, SEQ.ID.NO. 23, SEQ.ID.NO.25, and SEQ.ID.NO.27.
 6. The method of claim 1, wherein said B3 domain transcriptionfactors are selected from a group of polynucleotides consisting ofSEQ.ID.NO. 29, SEQ.ID.NO.31, SEQ.ID.NO. 33, SEQ.ID.NO. 35, SEQ.ID.NO.37, SEQ.ID.NO. 39, SEQ.ID.NO. 41, SEQ.ID.NO. 43, SEQ.ID.NO.45,SEQ.ID.NO. 47, SEQ.ID.NO. 49, SEQ.ID.NO.51, SEQ.ID.NO. 53, SEQ.ID.NO.55, SEQ.ID.NO. 57, SEQ.ID.NO. 59, SEQ.ID.NO. 61 SEQ.ID.NO. 63, andSEQ.ID.NO.65.
 7. The method of claim 1, wherein said transgenic plant isa monocot plant or a dicot plant.
 8. The method of claim 1, wherein saidexpression vector is a tissue-specific expression vector.
 9. A method ofproducing a transgenic plant comprising a) transforming a plant cellwith an expression vector comprising i) a B3-domain transcription factorhaving a polypeptide sequence with a B3 domain which is at least 40%identical to the B3 domain of SEQ. ID NO:2 or SEQ. ID. NO.: 4 and ii) abZIP transcription factor having a polypeptide sequence with a bZIPdomain which is at least 40% identical to the bZIP domain of SEQ. ID NO:6, and b) generating from the plant cell a transgenic plant with anincreased tolerance to environmental stress as compared to a wild typevariety of the plant.
 10. The method of claim 9, wherein said B3-domaintranscription factor is VP1 as defined by SEQ.ID.NO.:
 2. 11. The methodof claim 10, wherein said bZIP domain transcription factor is ABI5 asdefined by SEQ.ID.NO.: 6 or ABF1 as defined by SEQ.ID.NO.:
 8. 12. Themethod of claim 9, wherein said bZIP domain transcription factor is ABI5as defined by SEQ.ID.NO.: 6 or ABF1 as defined by SEQ.ID.NO.:
 8. 13. Themethod of claim 9, wherein said bZIP domain transcription factor isselected from a group of polypeptides consisting of SEQ.ID.NO. 10,SEQ.ID.NO. 12, SEQ.ID.NO. 16, SEQ.ID.NO. 18, SEQ.ID.NO.20, SEQ.ID.NO.22, SEQ.ID.NO. 24, SEQ.ID.NO.26, and SEQ.ID.NO.
 28. 14. The method ofclaim 9, wherein said B3 domain transcription factors are selected froma group of polypeptides consisting of SEQ.ID.NO. 30, SEQ.ID.NO.32,SEQ.ID.NO. 34, SEQ.ID.NO. 36, SEQ.ID.NO. 38, SEQ.ID.NO. 40, SEQ.ID.NO.42, SEQ.ID.NO. 44, SEQ.ID.NO.46, SEQ.ID.NO. 48, SEQ.ID.NO. 50,SEQ.ID.NO.52, SEQ.ID.NO. 54, SEQ.ID.NO. 56, SEQ.ID.NO. 58, SEQ.ID.NO.60, SEQ.ID.NO. 62 SEQ.ID.NO. 64, and SEQ.ID.NO.66.
 15. The method ofclaim 9, wherein said transgenic plant is a monocot plant or a dicotplant.
 16. The method of claim 9, wherein said expression vector is atissue-specific expression vector.
 17. A method of producing atransgenic plant comprising a) transforming a plant cell with anexpression vector comprising i) a first B3-domain transcription factorhaving a polynucleotide sequence with a B3 domain which is at least 25%identical to the B3 domain of SEQ. ID NO:1 and ii) a second B3-domaintranscription factor having a polynucleotide sequence with a B3 domainwhich is at least 25% identical to the B3 domain of SEQ. ID NO: 3, andb) generating from the plant cell a transgenic plant with an increasedtolerance to environmental stress as compared to a wild type variety ofthe plant.
 18. The method of claim 17, wherein said first B3 domaintranscription factor is VP1 as defined by SEQ. ID. NO.:
 1. 19. Themethod of claim 18, wherein said second B3 domain transcription factoris RAV2 as defined by SEQ. ID. NO.:
 3. 20. The method of claim 17,wherein said first B3 domain transcription factor is RAV2 as defined bySEQ. ID. NO.:
 3. 21. The method of claim 17, wherein said first B3domain transcription factor or said second B3 domain transcriptionfactor is selected from a group of polynucleotides consisting of SEQ.ID. NO.:
 29. SEQ. ID. NO.:31, SEQ. ID. NO.:33, SEQ. ID. NO.:35, SEQ. ID.NO.:37, SEQ. ID. NO.:39, SEQ. ID. NO.:41, SEQ. ID. NO.:43, SEQ. ID.NO.:45, SEQ. ID. NO.:47, SEQ. ID. NO.:49, SEQ. ID. NO.:51, SEQ. ID.NO.:53, SEQ. ID. NO.:55, SEQ. ID. NO.:57, SEQ. ID. NO.:59, SEQ. ID.NO.:61, SEQ. ID. NO.:63 and SEQ. ID. NO.:65.
 22. The method of claim 17,wherein said transgenic plant is a monocot plant or a dicot plant. 23.The method of claim 17, wherein said expression vector is atissue-specific expression vector.
 24. A method of producing atransgenic plant comprising a) transforming a plant cell with anexpression vector comprising i) a first B3-domain transcription factorhaving a polypeptide sequence with a B3 domain which is at least 40%identical to the B3 domain of SEQ. ID NO:2 and ii) a second B3-domaintranscription factor having a polypeptide sequence with a B3 domainwhich is at least 40% identical to the B3 domain of SEQ. ID NO: 4, andb) generating from the plant cell a transgenic plant with an increasedtolerance to environmental stress as compared to a wild type variety ofthe plant.
 25. The method of claim 24, wherein said first B3-domaintranscription factor is VP1 as defined by SEQ.ID.NO.:
 2. 26. The methodof claim 25, wherein said second B3-domain transcription factor is RAV2as defined by SEQ.ID.NO.:
 4. 27. The method of claim 24, wherein saidfirst B3 domain transcription factor is RAV2 as defined by SEQ. IS. NO.:4.
 28. The method of claims 24, wherein said first B3 domaintranscription factor or said second B3 domain transcription factor isselected from a group of polypeptides consisting of SEQ.ID.NO.: 30,SEQ.ID.NO.:32, SEQ.ID.NO.:34, SEQ.ID.NO.:36, SEQ. ID.NO.:38,SEQ.ID.NO.:40, SEQ.ID.NO.:42, SEQ.ID.NO.:44, SEQ.ID.NO.:46, SEQ.ID.NO.:48, SEQ.ID.NO.:50, SEQ.ID.NO.:52, SEQ.ID.NO.:54, SEQ.ID.NO.:56,SEQ. ID.NO.:58, SEQ.ID.NO.:60, SEQ.ID.NO.:62, SEQ.ID.NO.:64, andSEQ.ID.NO.:66.
 29. The method of claim 24, wherein said transgenic plantis a monocot plant or a dicot plant.
 30. The method of claim 24, whereinsaid expression vector is a tissue-specific expression vector.
 31. Atransgenic plant, comprising a) a B3-domain transcription factor havinga polynucleotide sequence with a B3 domain which is at least 25%identical to the B3 domain of SEQ. ID. NO: 1 or SEQ. ID. NO. 3 operablylinked to a tissue specific promoter, and b) a bZIP domain transcriptionfactor having a polynucleotide sequence with a bZIP domain which is atleast 25% identical to the bZIP domain of SEQ. ID. NO.:5 operably linkedto a tissue specific promoter, wherein expression of the B3-domaintranscription factor and the bZIP domain transcription factor in thetissue of said plant is effective to activate transcription of a geneoperably linked to a promoter with which said transcription factorinteracts.
 32. The transgenic plant of claim 31, wherein said B3-domaintranscription factor is VP1 as defined by SEQ.ID.NO.:
 1. 33. Thetransgenic plant of claim 32, wherein said bZIP domain transcriptionfactor is ABI5 as defined by SEQ.ID.NO.: 5 or ABF1 as defined bySEQ.ID.NO.:
 7. 34. The transgenic plant of claim 31, wherein said bZIPdomain transcription factor is ABI5 as defined by SEQ.ID.NO.: 5 or ABF1as defined by SEQ.ID.NO.:
 7. 35. The transgenic plant of claim 31,wherein said bZIP domain transcription factor is selected from a groupof polynucleotides consisting of SEQ.ID.NO. 9, SEQ.ID.NO. 11, SEQ.ID.NO.15, SEQ.ID.NO. 17, SEQ.ID.NO.19, SEQ.ID.NO. 21, SEQ.ID.NO. 23,SEQ.ID.NO.25, and SEQ.ID.NO.
 27. 36. The transgenic plant of claim 31,wherein said B3 domain transcription factors are selected from a groupof polynucleotides consisting of SEQ.ID.NO. 29, SEQ.ID.NO.31, SEQ.ID.NO.33, SEQ.ID.NO. 35, SEQ.ID.NO. 37, SEQ.ID.NO. 39, SEQ.ID.NO. 41,SEQ.ID.NO. 43, SEQ.ID.NO.45, SEQ.ID.NO. 47, SEQ.ID.NO. 49, SEQ.ID.NO.51,SEQ.ID.NO. 53, SEQ.ID.NO. 55, SEQ.ID.NO. 57, SEQ.ID.NO. 59, SEQ.ID.NO.61 SEQ.ID.NO. 63, and SEQ.ID.NO.65.
 37. The transgenic plant of claim31, wherein said transgenic plant is a monocot plant or a dicot plant.38. A transgenic plant, comprising a) a B3-domain transcription factorhaving a polypeptide sequence with a B3 domain which is at least 40%identical to the B3 domain of SEQ. ID. NO:2 or SEQ. ID. NO. 4 operablylinked to a tissue specific promoter, and b) a bZIP domain transcriptionfactor having a polypeptide sequence with a bZIP domain which is atleast 40% identical to the bZIP domain of SEQ. ID. NO.:6 operably linkedto a tissue specific promoter, wherein expression of the B3-domaintranscription factor and the bZIP domain transcription factor in thetissue of said plant is effective to activate transcription of a geneoperably linked to a promoter with which said transcription factorinteracts.
 39. The transgenic plant of claim 38, wherein said B3-domaintranscription factor is VP1 as defined by SEQ.ID.NO.:
 2. 40. Thetransgenic plant of claim 39, wherein said bZIP domain transcriptionfactor is ABI5 as defined by SEQ.ID.NO.: 6 or ABF1 as defined bySEQ.ID.NO.:
 8. 41. The transgenic plant of claim 38, wherein said bZIPdomain transcription factor is ABI5 as defined by SEQ.ID.NO.: 6 ABF1 asdefined by SEQ.ID.NO.:
 8. 42. The transgenic plant of claim 38, whereinsaid bZIP domain transcription factor is selected from a group ofpolypeptides consisting of SEQ.ID.NO. 10, SEQ.ID.NO. 12, SEQ.ID.NO. 16,SEQ.ID.NO. 18, SEQ.ID.NO.20, SEQ.ID.NO. 22, SEQ.ID.NO. 24, SEQ.ID.NO.26,and SEQ.ID.NO.
 28. 43. The transgenic plant of claim 38, wherein said B3domain transcription factors are selected from a group of polypeptidesconsisting of SEQ.ID.NO. 30, SEQ.ID.NO.32, SEQ.ID.NO. 34, SEQ.ID.NO. 36,SEQ.ID.NO. 38, SEQ.ID.NO. 40, SEQ.ID.NO. 42, SEQ.ID.NO. 44,SEQ.ID.NO.46, SEQ.ID.NO. 48, SEQ.ID.NO. 50, SEQ.ID.NO.52, SEQ.ID.NO. 54,SEQ.ID.NO. 56, SEQ.ID.NO. 58, SEQ.ID.NO. 60, SEQ.ID.NO. 62 SEQ.ID.NO.64, and SEQ.ID.NO.66.
 44. The transgenic plant of claim 38, wherein saidtransgenic plant is a monocot plant or a dicot plant.
 45. A transgenicplant, comprising a) a first B3-domain transcription factor having apolynucleotide sequence with a B3 domain which is at least 25% identicalto the B3 domain of SEQ. ID. NO: 1 operably linked to a tissue specificpromoter, and b) a second B3-domain domain transcription factor having apolynucleotide sequence with a B3 domain which is at least 25% identicalto the B3 domain of SEQ. ID. NO.:3 operably linked to a tissue specificpromoter, wherein expression of the first B3-domain transcription factorand the second B3 domain transcription factor in the tissue of saidplant is effective to activate transcription of a gene operably linkedto a promoter with which said transcription factor interacts.
 46. Thetransgenic plant of claim 45, wherein said first B3 domain transcriptionfactor is VP1 as defined by SEQ. ID. NO.:
 1. 47. The transgenic plant ofclaim 46, wherein said second B3 domain transcription factor is RAV2 asdefined by SEQ. ID. NO.:
 3. 48. The transgenic plant of claim 45,wherein said first B3 domain transcription factor is RAV2 as defined bySEQ. ID. NO.:
 3. 49. The transgenic plant of claim 45, wherein saidfirst B3 domain transcription factor or said second B3 domaintranscription factor is selected from a group of polynucleotidesconsisting of SEQ. ID. NO.:
 29. SEQ. ID. NO.:31, SEQ. ID. NO.:33, SEQ.ID. NO.:35, SEQ. ID. NO.:37, SEQ. ID. NO.:39, SEQ. ID. NO.:41, SEQ. ID.NO.:43, SEQ. ID. NO.:45, SEQ. ID. NO.:47, SEQ. ID. NO.:49, SEQ. ID.NO.:51, SEQ. ID. NO.:53, SEQ. ID. NO.:55, SEQ. ID. NO.:57, SEQ. ID.NO.:59, SEQ. ID. NO.:61, SEQ. ID. NO.:63 and SEQ. ID. NO.:65.
 50. Thetransgenic plant of claim 45, wherein said transgenic plant is a monocotplant or a dicot plant.
 51. A transgenic plant, comprising a) a firstB3-domain transcription factor having a polypeptide sequence with a B3domain which is at least 40% identical to the B3 domain of SEQ. ID. NO:2operably linked to a tissue specific promoter, and b) a second B3-domaindomain transcription factor having a polypeptide sequence with a B3domain which is at least 40% identical to the B3 domain of SEQ. ID.NO.:4 operably linked to a tissue specific promoter, wherein expressionof the first B3-domain transcription factor and the second B3 domaintranscription factor in the tissue of said plant is effective toactivate transcription of a gene operably linked to a promoter withwhich said transcription factor interacts.
 52. The transgenic plant ofclaim 51, wherein said first B3-domain transcription factor is VP1 asdefined by SEQ.ID.NO.:
 2. 53. The transgenic plant of claim 52, whereinsaid second B3-domain transcription factor is RAV2 as defined bySEQ.ID.NO.:
 4. 54. The transgenic plant of claim 51, wherein said firstB3 domain transcription factor is RAV2 as defined by SEQ. IS. NO.: 4.55. The transgenic plant of claims 51, wherein said first B3 domaintranscription factor or said second B3 domain transcription factor isselected from a group of polypeptides consisting of SEQ.ID.NO.: 30,SEQ.ID.NO.:32, SEQ.ID.NO.:34, SEQ.ID.NO.:36, SEQ. ID.NO.:38,SEQ.ID.NO.:40, SEQ.ID.NO.:42, SEQ.ID.NO.:44, SEQ.ID.NO.:46, SEQ.ID.NO.:48, SEQ.ID.NO.:50, SEQ.ID.NO.:52, SEQ.ID.NO.:54, SEQ.ID.NO.:56,SEQ. ID.NO.:58, SEQ.ID.NO.:60, SEQ.ID.NO.:62, SEQ.ID.NO.:64, andSEQ.ID.NO.:66.
 56. The transgenic plant of claim 51, wherein saidtransgenic plant is a monocot plant or a dicot plant.
 57. A method ofscreening for a polynucleotide which encodes a B3 domain protein or abZIP domain protein comprising, a) hybridizing a polynucleotide havingat least 25% identical to the B3 domain of SEQ. ID. NO:1 or SEQ. ID.NO.: 3 or a polynucleotide having at least 25% identity to the bZIPdomain of SEQ. ID. NO. 5: to the polynucleotide to be screened; b)expressing the polynucleotide to produce a protein; and c) detecting thepresence or absence of B3 domain activity or bZIP domain activity insaid protein.
 58. A vector comprising an isolated polynucleotide havinga polynucleotide sequence with a B3 domain which is at least 25%identical to the B3 domain of SEQ. ID. NO. 1 or SEQ. ID. NO.: 3 and anisolated polynucleotide having a polynucleotide sequence with a bZIPdomain which is at least 25% identical to the bZIP domain of SEQ. ID.NO.:
 5. 59. The vector of claim 58, wherein said B3-domain transcriptionfactor is VP1 as defined by SEQ.ID.NO.:
 1. 60. The vector of claim 59,wherein said bZIP domain transcription factor is selected from a groupof polynucleotides consisting of SEQ. ID. NO. 5, and SEQ. ID. NO.
 7. 61.The vector of claim 58, wherein said bZIP domain transcription factor isselected from a group of polynucleotides consisting of SEQ. ID. NO. 5.and SEQ. ID. NO.
 7. 62. The vector of claim 58 wherein said bZIP domaintranscription factor is selected from a group of polynucleotidesconsisting of SEQ. ID. NO. 9, SEQ. ID. NO. 11, SEQ. ID. NO. 15, SEQ. ID.NO. 17, SEQ. ID. NO. 19, SEQ. ID. NO. 21, SEQ. ID. NO. 23, SEQ. ID. NO.25 and SEQ. ID. NO.
 27. 63. The vector of claim 58 wherein said B3domain transcription factor is selected from a group of polynucleotidesconsisting of SEQ. ID. NO. 29, SEQ. ID. NO. 31, SEQ. ID. NO. 33, SEQ.ID. NO. 35, SEQ. ID. NO. 37, SEQ. ID. NO. 39, SEQ. ID. NO. 41, SEQ. ID.NO. 43, SEQ. ID. NO. 45, SEQ. ID. NO. 47, SEQ. ID. NO. 49, SEQ. ID. NO.51, SEQ. ID. NO. 53, SEQ. ID. NO. 55, SEQ. ID. NO. 57, SEQ. ID. NO. 59,SEQ. ID. NO. 61, SEQ. ID. NO. 63, and SEQ. ID. NO.
 65. 64. A host cellcomprising an isolated polynucleotide having a polynucleotide sequencewith a B3 domain which is at least 25% identical to the B3 domain ofSEQ. ID. NO. 1 or SEQ. ID. NO.: 3 and an isolated polynucleotide havinga polynucleotide sequence with a bZIP domain which is at least 25%identical to the bZIP domain of SEQ. ID. NO.:
 5. 65. The host cell ofclaim 64, wherein said B3-domain transcription factor is VP1 as definedby SEQ.ID.NO.:1.
 66. The host cell of claim 65, wherein said bZIP domaintranscription factor is selected from a group of polynucleotidesconsisting of SEQ. ID. NO.
 5. and SEQ. ID. NO.
 7. 67. The host cell ofclaim 64, wherein said bZIP domain transcription factor is selected froma group of polynucleotides consisting of SEQ. ID. NO.
 5. and SEQ. ID.NO.
 7. 68. The host cell of claim 64 wherein said bZIP domaintranscription factor is selected from a group of polynucleotidesconsisting of SEQ. ID. NO. 9, SEQ. ID. NO. 11, SEQ. ID. NO. 15, SEQ. ID.NO. 17, SEQ. ID. NO. 19, SEQ. ID. NO. 21, SEQ. ID. NO. 23, SEQ. ID. NO.25 and SEQ. ID. NO.
 27. 69. The host cell of claim 64 wherein said B3domain transcription factor is selected from a group of polynucleotidesconsisting of SEQ. ID. NO. 29, SEQ. ID. NO. 31, SEQ. ID. NO. 33, SEQ.ID. NO. 35, SEQ. ID. NO. 37, SEQ. ID. NO. 39, SEQ. ID. NO. 41, SEQ. ID.NO. 43, SEQ. ID. NO. 45, SEQ. ID. NO. 47, SEQ. ID. NO. 49, SEQ. ID. NO.51, SEQ. ID. NO. 53, SEQ. ID. NO. 55, SEQ. ID. NO. 57, SEQ. ID. NO. 59,SEQ. ID. NO. 61, SEQ. ID. NO. 63, and SEQ. ID. NO.
 65. 70. A vectorcomprising a first isolated polynucleotide having a polynucleotidesequence with a B3 domain which is at least 25% identical to the B3domain of SEQ. ID. NO. 1 and a second isolated polynucleotide having apolynucleotide sequence with a B3 domain which is at least 25% identicalto the B3 domain of SEQ. ID. NO.:
 3. 71. The vector of claim 70, whereinsaid first isolated polynucleotide is VP1 as defined by SEQ. ID. NO. 1.72. The vector of claim 71, wherein said second isolated polynucleotideis RAV2 as defined by SEQ. ID. NO.:
 3. 73. The vector of claim 70,wherein said second isolated polynucleotide is RAV2 as defined by SEQ.ID. NO.:
 3. 74. The vector of claim 70, wherein said first isolatedpolynucleotide or said second isolated polynucleotide is selected from agroup of polynucleotides consisting of SEQ. ID. NO.: 29, SEQ. ID.NO.:31, SEQ. ID. NO.:33, SEQ. ID. NO.:35, SEQ. ID. NO.:37, SEQ. ID.NO.:39, SEQ. ID. NO.:41, SEQ. ID. NO.:43, SEQ. ID. NO.:45, SEQ. ID.NO.:47, SEQ. ID. NO.:49, SEQ. ID. NO.:51, SEQ. ID. NO.:53, SEQ. ID.NO.:55, SEQ. ID. NO.: 57, SEQ. ID. NO.:59, SEQ. ID. NO.:61, SEQ. ID.NO.:63, and SEQ. ID. NO.:65.
 75. A host cell comprising a first isolatedpolynucleotide having a polynucleotide sequence with a B3 domain whichis at least 25% identical to the B3 domain of SEQ. ID. NO. 1 and asecond isolated polynucleotide having a polynucleotide sequence with aB3 domain which is at least 25% identical to the B3 domain of SEQ. ID.NO.:
 3. 76. The vector of claim 75, wherein said first isolatedpolynucleotide is VP1 as defined by SEQ. ID. NO.
 1. 77. The vector ofclaim 76, wherein said second isolated polynucleotide is RAV2 as definedby SEQ. ID. NO.:
 3. 78. The vector of claim 75, wherein said secondisolated polynucleotide is RAV2 as defined by SEQ. ID. NO.:
 3. 79. Thevector of claim 75, wherein said first isolated polynucleotide or saidsecond isolated polynucleotide is selected from a group ofpolynucleotides consisting of SEQ. ID. NO.: 29, SEQ. ID. NO.:31, SEQ.ID. NO.:33, SEQ. ID. NO.:35, SEQ. ID. NO.:37, SEQ. ID. NO.:39, SEQ. ID.NO.:41, SEQ. ID. NO.:43, SEQ. ID. NO.:45, SEQ. ID. NO.:47, SEQ. ID.NO.:49, SEQ. ID. NO.:51, SEQ. ID. NO.:53, SEQ. ID. NO.:55, SEQ. ID. NO.:57, SEQ. ID. NO.:59, SEQ. ID. NO.:61, SEQ. ID. NO.:63, and SEQ. ID.NO.:65.